Prodrugs and methods of use thereof

ABSTRACT

The invention relates to compounds of use as targeted cytotoxic agents and methods of use thereof. In particular, the invention relates to prodrugs that are substantially resistant to human AKR1C3 enzyme metabolism, methods of cell ablation using said compounds and methods of treatment of cancer and other hyperproliferative disorders using said compounds.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a continuation-in-part of U.S. application Ser. No. 15/339,041,filed Oct. 31, 2016, which is a continuation of U.S. application Ser.No. 14/423,359, filed on Feb. 23, 2015 as the U.S. national phase ofPCT/NZ2013/000150, filed on Aug. 22, 2013, which claims the benefit ofNew Zealand Application No. 602004, filed Aug. 23, 2012, all of whichare incorporated by reference herein in their entireties.

FIELD OF THE INVENTION

The invention relates to compounds of use as targeted cytotoxic agentsand methods of use thereof. In particular embodiments, the inventionrelates to nitrobenzamide mustards, nitrobenzamide mustard alcohols andtheir corresponding phosphate esters.

BACKGROUND OF THE INVENTION

The use of tumour-selective prodrugs (relatively inactive compounds thatcan be selectively converted to more active compounds in vivo) is avaluable concept in cancer therapy (see for example, Denny, Eur. J: Med.Chem. (2001) 36,577). For example a prodrug may be converted into ananti-tumour agent under the influence of an enzyme that is linkable to amonoclonal antibody that will bind to a tumour associated antigen. Thecombination of such a prodrug with such an enzyme/monoclonal antibodyconjugate represents a very powerful clinical agent. This approach tocancer therapy, often referred to as “antibody directed enzyme prodrugtherapy” (ADEPT), is disclosed in international publicationWO/1988/007378.

A further therapeutic approach termed “virus-directed enzyme prodrugtherapy” (VDEPT) has been proposed as a method for treating tumour cellsin patients using prodrugs. Tumour cells are targeted with a viralvector carrying a gene encoding an enzyme capable of activating aprodrug. The gene may be transcriptionally regulated by tissue specificpromoter or enhancer sequences. The viral vector enters tumour cells andexpresses the enzyme, in order that a prodrug is converted to an activedrug within the tumour cells (Huber et al., Proc. Natl. Acad. Sci. USA(1991) 88, 8039). Alternatively, non-viral methods for the delivery ofgenes have been used. Such methods include calcium phosphateco-precipitation, microinjection, liposomes, direct DNA uptake, andreceptor mediated DNA-transfer. These are reviewed in Morgan & French,Annu. Rev. Bioehem., 1993, 62; 191. The term “GDEPT” (gene-directedenzyme prodrug therapy) is used to include both viral and non-viraldelivery systems (Denny et al U.S. Pat. No. 6,310,237). One example of anon-viral delivery system being the tumour colonising bacteriaClostridia, utilised in an approach termed clostridia-directed enzymeprodrug therapy (CDEPT).

Many nitroaromatic compounds can be reduced by both mammalian andbacterial flavoprotein enzymes, which effect stepwise addition of up tosix electrons. The major enzymatic metabolite is usually the 4-electronreduced species (hydroxylamine). A number of nitrophenyl mustards andnitrophenylaziridines have been reported as prodrugs for use ingene-directed enzyme prodrug therapy (GDEPT) in conjunction withnitroreductase enzymes. In particular, CB 1954[5-(aziridin-1-yl)-2,4-dinitrobenzamide] (compound 1, scheme 1) isreported to be a substrate for the aerobic bacterial nitroreductase NTR(nfsB gene product) isolated from E. coli (Boland et al., Biochem.Pharmacol. 1991, 41, 867-875; Anlezark et al., Biochem. Pharmacol, 15,1992, 44, 2289-2295; Parkinson et al., J. Med. Chem. 2000, 43, 3624).This compound has been used as a prodrug in both ADEPT (Knox et al.,Biochem. Pharmacol., 1995, 49, 1641-1647) and GDEPT (Bridgewater et al.,Eur. J. Cancer, 1995, 31A, 2362-2370; Bailey et al., Gene Ther., 1996,3, 1143-1150; Bailey and Hart, Gene Ther., 1997, 4, 80-81; Green et al.,Cancer Gene Ther., 1997, 4, 229-238) applications, including a clinicaltrial (Chung-Faye et al., Clin. Cancer Res., 2001, 7, 2662-2668).Similarly, the dinitrophenyl mustard SN 23862 (compound 2, scheme 1) isalso a substrate for E. coli NfsB, and shows selective toxicity towardscell lines that express the enzyme. It is activated by nitro groupreduction (Palmer et al., J. Med. Chem., 1995, 38, 1229; Kestell et al.,Cancer Chemother. Pharmacol., 2000, 46, 365-374). The 4-SO₂Me derivative(compound 3, scheme 1) was also a substrate (Atwell et al., Anti-CancerDrug Des., 1996, 11, 553), as was the dibromo mustard analogue (compound4, scheme 1) (Atwell et al., J. Med. Chem., 2007, 50, 1197-1212).Prodrugs 1-4 (scheme 1) have poor aqueous solubility. For example, todetermine the efficacy of prodrug 4 in xenograft-bearing nude mice, itwas administered in either neat DMSO or DMSO/polyethylene glycol/water(Atwell et al., J. Med. Chem., 2007, 50, 1197-1212) resulting in a largevariations in maximum tolerated dose.

Scheme 1

Some phosphate analogues of mustards have been described, for thepurpose of solubilising the compounds. The best known is estramustinephosphate, which has been shown to bind to tubulin binding domains onvarious microtubule-associated proteins (Moraga et al., Biochim.Biophys. Acta, 1992, 1 121, 97-103), and which has been shown to beactive in advanced breast cancer (Keren-Rosenberg et al., Semin. Oncol.,1997, 24(Suppl. 3), 26-29), but has not been shown to be activated byNTR.

Dinitrobenzamide mustards bearing alcohol side chains pendant at acarboxamide (—CONH—) group and their phosphate derivatives are describedas bioreductive drugs for GDEPT applications (WO/2008/030112 andWO/2005/042471). Central to the disclosure are prodrugs that providecell ablation with substantially minimal bystander effect, a term usedto describe the back diffusion of cytotoxic metabolites from bacterialnitroreductase-expressing target cells to ablate bacterialnitroreductase naive cells. No bystander efficiency data is provided.

The ability to sterilise neighbouring cells otherwise unable to activatethe targeted cytotoxic agent is of central importance to the activity ofthe agents in combination with nitroreductase enzymes. Gene/enzymedelivery technologies utilised in approaches such as GDEPT, VDEPT, CDEPTand ADEPT are inherently heterogeneous, necessitating efficientredistribution of activated cytotoxic metabolites to inhibit a largerpopulation of neighbouring cells. Thus the bystander effect is animportant mechanism to compensate for this anticipated heterogeneity bygenerating cytotoxic metabolites that diffuse locally to ablateneighbouring vector-naïve cells.

In addition to activation by exogenous oxygen-independent two-electronnitroreductases it is desirable to design nitroaromatic prodrugs,bearing a nitro substituent of an appropriate electron affinity that itis able to be reduced by endogenous human one-electron reductases toproduce a nitro radical anion that can be readily back-oxidised bymolecular oxygen. In well-oxygenated tissues in the body the parentprodrug is re-formed in a futile redox cycle, however in the presence ofpathological hypoxia found in human solid tumours, net reduction tohydroxylamine and amine cytotoxic metabolites is able to occur providingtumour-selective cell killing. Such compounds are termedhypoxia-activated prodrugs (HAP) or hypoxia-selective cytotoxins (HSC).

The Phase II clinical candidate PR-104 is a 3,5-dinitrobenzamidewater-soluble phosphate pre-prodrug that, following hydrolysis bysystemic phosphatases, releases the ‘hypoxia-activated’ and ‘bacterialnitroreductase-activated’ prodrug PR-104A. Metabolism of PR-104A byendogenous human one-electron reductases in hypoxic cells of a tumour orby exogenous oxygen-independent two-electron nitroreductases, such asbacterial nitroreductases genetically engineered to be expressed in atumour, produces the DNA crosslinking mustard cytotoxic metabolitesPR-104H and PR-104M (Scheme 2) (Patterson et al., Clin Can Res 2007,13:3922-32).

Unexpectedly PR-104A is also subject to 2e-reduction by an endogenoushuman reductase called aldo-ketoreductase 1C3 (AKR1C3). This aerobicpathway yields identical cytotoxic metabolites. Expression of AKR1C3 inhuman CD34⁺ myeloid progenitor cells may result in a lack of selectivityof PR-104 for solid tumours versus normal bone marrow, compromisingPR-104's therapeutic index. It is desirable therefore to eliminate thisoff-mechanism aerobic activation of PR-104 by AKR1C3.

It is an object of the present invention to provide one or more prodrugsthat are substantially free of activation by human AKR1C3 enzyme, or atleast to provide the public with a useful choice.

SUMMARY OF THE INVENTION

In a first aspect of the invention there is provided a compound ofFormula (I)

wherein W represents Cl, Br, I, OSO₂R,

X represents Cl, Br, I, OSO₂R,

Y represents H, CN, SO₂R,

each R independently represents a lower C₁₋₆ alkyl group,

Z is selected from any of the radicals of Formula (Ia)

-   -   where        -   R₁ represents H, or a lower C₁₋₆ alkyl group;        -   R2 and R3 may independently represent H, or a lower C₁₋₆            alkyl group, or        -   R2 and R3 together may be linked to form a substituted or            unsubstituted heterocyclic ring comprising 5 or 6 members;        -   n represents 2 to 6;        -   * represents a point of attachment to Formula I;

or a pharmaceutically acceptable salt of said compound.

In a particular embodiment of the first aspect, the invention provides acompound of Formula (Ib)

wherein Y represents H, CN, SO₂R,

R represents a methyl or ethyl group,

Z is selected from any of the radicals of Formula (Ic)

-   -   where        -   R₁ represents H, or a lower C₁₋₆ alkyl group;        -   R₂ and R₃ may independently represent H, or a lower C1-6            alkyl group, or        -   R₂ and R₃ together may be linked to form a substituted or            unsubstituted heterocyclic ring comprising 5 or 6 members;        -   n represents 2 to 6;        -   * represents a point of attachment to Formula Ib;

or a pharmaceutically acceptable salt of said compound.

In one embodiment the compound of Formula (I) comprises a compoundrepresented by formula (Id),

wherein n represents 2 to 6,

W represents Cl, Br, I, OSO₂R,

X represents Cl, Br, I, OSO₂R,

each R independently represents a lower C₁₋₆ alkyl group, and

R₁ represents H, or a lower C₁₋₆ alkyl group.

In another embodiment W is bromine or iodine.

In another embodiment X is bromine or OSO₂Me.

In another embodiment R is methyl or ethyl.

In another embodiment R₁ is hydrogen, methyl or ethyl.

In another embodiment n represents 2 or 3

In a particular embodiment, the compound of Formula (I) comprises acompound represented by formula (Ie),

wherein n represents 2 to 6, and

R represents methyl or ethyl.

In another embodiment the compound of Formula (I) is selected from:

-   2-(5-(bis(2-bromoethyl)amino)-N-methyl-4-(methylsulfonyl)-2-nitrobenzamido)ethyl    dihydrogen phosphate (compound 10),-   2-(5-(bis(2-bromoethyl)amino)-4-(ethylsulfonyl)-N-methyl-2-nitrobenzamido)ethyl    dihydrogen phosphate (compound 11),-   3-(5-(bis(2-bromoethyl)amino)-N-methyl-4-(methylsulfonyl)-2-nitrobenzamido)propyl    dihydrogen phosphate (compound 12),-   3-(5-(bis(2-bromoethyl)amino)-4-(ethylsulfonyl)-N-methyl-2-nitrobenzamido)propyl    dihydrogen phosphate (compound 13),-   2-(5-(bis(2-bromoethyl)amino)-4-cyano-N-methyl-2-nitrobenzamido)ethyl    dihydrogen phosphate (compound 69),-   3-(5-(bis(2-bromoethyl)amino)-4-cyano-N-methyl-2-nitrobenzamido)propyl    dihydrogen phosphate (compound 70),-   2-(5-(bis(2-bromoethyl)amino)-4-(methylsulfonyl)-2-nitrobenzamido)ethyl    dihydrogen phosphate (compound 300),-   3-(5-(bis(2-bromoethyl)amino)-4-(methylsulfonyl)-2-nitrobenzamido)propyl    dihydrogen phosphate (compound 308),-   3-(5-(bis(2-bromoethyl)amino)-4-(ethylsulfonyl)-2-nitrobenzamido)propyl    dihydrogen phosphate (compound 309),-   2-((2-bromoethyl)(2-cyano-5-(methyl(2-(phosphonooxy)ethyl)carbamoyl)-4-nitrophenyl)amino)ethyl    methanesulfonate (compound 350), and-   2-((2-bromoethyl)(2-cyano-5-(methyl(3-(phosphonooxy)propyl)carbamoyl)-4-nitrophenyl)amino)ethyl    methanesulfonate (compound 351).

In another embodiment the compound of Formula (I) is selected from acompound represented by formula (If),

wherein n represents 2 to 6,

W represents Cl, Br, I, OSO₂R,

X represents Cl, Br, I, OSO₂R,

each R independently represents a lower C₁₋₆ alkyl group, and

R₁ represents H, or a lower C₁₋₆ alkyl group.

In another embodiment W is bromine or iodine.

In another embodiment X is bromine or OSO₂Me.

In another embodiment R is methyl or ethyl.

In another embodiment R₁ is hydrogen, methyl or ethyl.

In another embodiment n represents 2 or 3

In particular embodiment, the compound of Formula (I) is selected from acompound represented by formula (Ig),

wherein n represents 2 to 6,

R represents methyl or ethyl.

In another embodiment the compound of Formula (I) is selected from:

-   5-(bis(2-bromoethyl)amino)-N-(2-hydroxyethyl)-N-methyl-4-(methylsulfonyl)-2-nitrobenzamide    (compound 14),-   5-(bis(2-bromoethyl)amino)-N-(3-hydroxypropyl)-N-methyl-4-(methylsulfonyl)-2-nitrobenzamide    (compound 17),-   5-(bis(2-bromoethyl)amino)-4-(ethylsulfonyl)-N-(2-hydroxyethyl)-N-methyl-2-nitrobenzamide    (compound 18),-   5-(bis(2-bromoethyl)amino)-4-cyano-N-(2-hydroxyethyl)-N-methyl-2-nitrobenzamide    (compound 67),-   5-(bis(2-bromoethyl)amino)-4-cyano-N-(3-hydroxypropyl)-N-methyl-2-nitrobenzamide    (compound 68),-   5-(bis(2-bromoethyl)amino)-N-(2-hydroxyethyl)-4-(methylsulfonyl)-2-nitrobenzamide    (compound 301),-   5-(bis(2-bromoethyl)amino)-4-(ethylsulfonyl)-N-(3-hydroxypropyl)-N-methyl-2-nitrobenzamide    (compound 305),-   5-(bis(2-bromoethyl)amino)-N-(3-hydroxypropyl)-4-(methylsulfonyl)-2-nitrobenzamide    (compound 306),-   5-(bis(2-bromoethyl)amino)-4-(ethylsulfonyl)-N-(3-hydroxypropyl)-2-nitrobenzamide    (compound 307),-   2-((2-bromoethyl)(2-cyano-5-((2-hydroxyethyl)(methyl)carbamoyl)-4-nitrophenyl)amino)ethyl    methanesulfonate (compound 348), and-   2-((2-bromoethyl)(2-cyano-5-((3-hydroxypropyl)(methyl)carbamoyl)-4-nitrophenyl)amino)ethyl    methanesulfonate (compound 349).

In another embodiment the compound of Formula (I) is selected from acompound represented by formula (Ih),

wherein W represents Cl, Br, I, OSO₂R,

X represents Cl, Br, I, OSO₂R,

each R independently represents a lower C₁₋₆ alkyl group,

R₁ represents H, or a lower C₁₋₆ alkyl group.

In another embodiment W is bromine or iodine.

In another embodiment X is bromine or OSO₂Me.

In another embodiment R is methyl or ethyl.

In another embodiment R₁ is methyl, ethyl, propyl or iso-propyl.

In another embodiment the compound of Formula (I) is selected from acompound represented by formula (Ii),

wherein R represents a lower C₁₋₆ alkyl group,

R₁ represents H, or a lower C₁₋₆ alkyl group.

In another embodiment R is methyl or ethyl.

In another embodiment R₁ is methyl or ethyl.

In another embodiment the compound of Formula (I) defined above isselected from:

-   (5-(bis(2-bromoethyl)amino)-4-(methylsulfonyl)-2-nitrophenyl)(4-methylpiperazin-1-yl)methanone    (compound 22),-   (5-(bis(2-bromoethyl)amino)-4-(methylsulfonyl)-2-nitrophenyl)(4-ethylpiperazin-1-yl)methanone    (compound 23),-   (5-(bis(2-bromoethyl)amino)-4-(methylsulfonyl)-2-nitrophenyl)(4-isopropylpiperazin-1-yl)methanone    (compound 24),-   (5-(bis(2-bromoethyl)amino)-4-(ethylsulfonyl)-2-nitrophenyl)(4-methylpiperazin-1-yl)methanone    (compound 25),-   (5-(bis(2-bromoethyl)amino)-4-(ethylsulfonyl)-2-nitrophenyl)(4-ethylpiperazin-1-yl)methanone    (compound 26),-   (5-(bis(2-bromoethyl)amino)-4-(ethylsulfonyl)-2-nitrophenyl)(4-isopropylpiperazin-1-yl)methanone    (compound 27),-   5-(bis(2-bromoethyl)amino)-4-(methylsulfonyl)-N-(2-morpholinoethyl)-2-nitrobenzamide    (compound 28),-   5-(bis(2-bromoethyl)amino)-N-methyl-4-(methylsulfonyl)-N-(2-morpholinoethyl)-2-nitrobenzamide    (compound 29),-   5-(bis(2-bromoethyl)amino)-4-(methylsulfonyl)-N-(3-morpholinopropyl)-2-nitrobenzamide    (compound 30),-   5-(bis(2-bromoethyl)amino)-N-methyl-4-(methylsulfonyl)-N-(3-morpholinopropyl)-2-nitrobenzamide    (compound 31),-   5-(bis(2-bromoethyl)amino)-N-(2-(4-methyl    piperazin-1-yl)ethyl)-4-(methylsulfonyl)-2-nitrobenzamide (compound    32),-   5-(bis(2-bromoethyl)amino)-N-methyl-N-(2-(4-methylpiperazin-1-yl)ethyl)-4-(methylsulfonyl)-2-nitrobenzamide    (compound 33),-   5-(bis(2-bromoethyl)amino)-N-(3-(4-methylpiperazin-1-yl)propyl)-4-(methylsulfonyl)-2-nitrobenzamide    (compound 34),-   5-(bis(2-bromoethyl)amino)-N-methyl-N-(3-(4-methylpiperazin-1-yl)propyl)-4-(methylsulfonyl)-2-nitrobenzamide    (compound 35),-   5-(bis(2-bromoethyl)amino)-N-(2-(dimethylamino)ethyl)-4-(methylsulfonyl)-2-nitrobenzamide    (compound 36),-   5-(bis(2-bromoethyl)amino)-N-(2-(dimethylamino)ethyl)-N-methyl-4-(methylsulfonyl)-2-nitrobenzamide    (compound 37),-   5-(bis(2-bromoethyl)amino)-N-(3-(dimethylamino)propyl)-4-(methylsulfonyl)-2-nitrobenzamide    (compound 38),-   5-(bis(2-bromoethyl)amino)-N-(3-(dimethylamino)propyl)-N-methyl-4-(methylsulfonyl)-2-nitrobenzamide    (compound 39),-   5-(bis(2-bromoethyl)amino)-4-(ethylsulfonyl)-N-(2-morpholinoethyl)-2-nitrobenzamide    (compound 40),-   5-(bis(2-bromoethyl)amino)-4-(ethylsulfonyl)-N-methyl-N-(2-morpholinoethyl)-2-nitrobenzamide    (compound 41),-   5-(bis(2-bromoethyl)amino)-4-(ethylsulfonyl)-N-(3-morpholinopropyl)-2-nitrobenzamide    (compound 42),-   5-(bis(2-bromoethyl)amino)-4-(ethylsulfonyl)-N-methyl-N-(3-morpholinopropyl)-2-nitrobenzamide    (compound 43),-   5-(bis(2-bromoethyl)amino)-4-(ethylsulfonyl)-N-(2-(4-methylpiperazin-1-yl)ethyl)-2-nitrobenzamide    (compound 44),-   5-(bis(2-bromoethyl)amino)-4-(ethylsulfonyl)-N-methyl-N-(2-(4-methylpiperazin-1-yl)ethyl)-2-nitrobenzamide    (compound 45),-   5-(bis(2-bromoethyl)amino)-4-(ethylsulfonyl)-N-(3-(4-methylpiperazin-1-yl)propyl)-2-nitrobenzamide    (compound 46),-   5-(bis(2-bromoethyl)amino)-4-(ethylsulfonyl)-N-methyl-N-(3-(4-methylpiperazin-1-yl)propyl)-2-nitrobenzamide    (compound 47),-   5-(bis(2-bromoethyl)amino)-N-(2-(dimethylamino)ethyl)-4-(ethylsulfonyl)-2-nitrobenzamide    (compound 48),-   5-(bis(2-bromoethyl)amino)-N-(2-(dimethylamino)ethyl)-4-(ethylsulfonyl)-N-methyl-2-nitrobenzamide    (compound 49),-   5-(bis(2-bromoethyl)amino)-N-(3-(dimethylamino)propyl)-4-(ethylsulfonyl)-2-nitrobenzamide    (compound 50),-   5-(bis(2-bromoethyl)amino)-N-(3-(dimethylamino)propyl)-4-(ethylsulfonyl)-N-methyl-2-nitrobenzamide    (compound 51),-   3-(5-(bis(2-bromoethyl)amino)-4-(methylsulfonyl)-2-nitrobenzamido)propanoic    acid (compound 52),-   4-(5-(bis(2-bromoethyl)amino)-4-(methylsulfonyl)-2-nitrobenzamido)butanoic    acid (compound 53),-   3-(5-(bis(2-bromoethyl)amino)-N-methyl-4-(methylsulfonyl)-2-nitrobenzamido)propanoic    acid (compound 54),-   4-(5-(bis(2-bromoethyl)amino)-N-methyl-4-(methylsulfonyl)-2-nitrobenzamido)butanoic    acid (compound 55),-   3-(5-(bis(2-bromoethyl)amino)-4-(ethylsulfonyl)-2-nitrobenzamido)propanoic    acid (compound 56),-   4-(5-(bis(2-bromoethyl)amino)-4-(ethylsulfonyl)-2-nitrobenzamido)butanoic    acid (compound 57),-   3-(5-(bis(2-bromoethyl)amino)-4-(ethylsulfonyl)-N-methyl-2-nitrobenzamido)propanoic    acid (compound 58),-   4-(5-(bis(2-bromoethyl)amino)-4-(ethylsulfonyl)-N-methyl-2-nitrobenzamido)butanoic    acid (compound 59)-   2-(bis(2-bromoethyl)amino)-4-(4-methylpiperazine-1-carbonyl)-5-nitrobenzonitrile    (compound 71),-   2-(bis(2-bromoethyl)amino)-4-(4-ethylpiperazine-1-carbonyl)-5-nitrobenzonitrile    (compound 72),-   2-(bis(2-bromoethyl)amino)-4-(4-isopropylpiperazine-1-carbonyl)-5-nitrobenzonitrile    (compound 73),-   3-(5-(bis(2-bromoethyl)amino)-4-cyano-2-nitrobenzamido)propanoic    acid (compound 74),-   4-(5-(bis(2-bromoethyl)amino)-4-cyano-2-nitrobenzamido)butanoic acid    (compound 75),-   3-(5-(bis(2-bromoethyl)amino)-4-cyano-N-methyl-2-nitrobenzamido)propanoic    acid (compound 76),-   4-(5-(bis(2-bromoethyl)amino)-4-cyano-N-methyl-2-nitrobenzamido)butanoic    acid (compound 77),-   5-(bis(2-bromoethyl)amino)-4-cyano-N-(2-morpholinoethyl)-2-nitrobenzamide    (compound 78),-   5-(bis(2-bromoethyl)amino)-4-cyano-N-methyl-N-(2-morpholinoethyl)-2-nitrobenzamide    (compound 79),-   5-(bis(2-bromoethyl)amino)-4-cyano-N-(3-morpholinopropyl)-2-nitrobenzamide    (compound 80),-   5-(bis(2-bromoethyl)amino)-4-cyano-N-methyl-N-(3-morpholinopropyl)-2-nitrobenzamide    (compound 81),-   5-(bis(2-bromoethyl)amino)-4-cyano-N-(2-(4-methyl    piperazin-1-yl)ethyl)-2-nitrobenzamide (compound 82),-   5-(bis(2-bromoethyl)amino)-4-cyano-N-methyl-N-(2-(4-methyl    piperazin-1-yl)ethyl)-2-nitrobenzamide (compound 83),-   5-(bis(2-bromoethyl)amino)-4-cyano-N-(3-(4-methyl    piperazin-1-yl)propyl)-2-nitrobenzamide (compound 84),-   5-(bis(2-bromoethyl)amino)-4-cyano-N-methyl-N-(3-(4-methyl    piperazin-1-yl)propyl)-2-nitrobenzamide (compound 85),-   5-(bis(2-bromoethyl)amino)-4-cyano-N-(2-(dimethylamino)ethyl)-2-nitrobenzamide    (compound 86),-   5-(bis(2-bromoethyl)amino)-4-cyano-N-(2-(dimethylamino)ethyl)-N-methyl-2-nitrobenzamide    (compound 87),-   5-(bis(2-bromoethyl)amino)-4-cyano-N-(3-(dimethylamino)propyl)-2-nitrobenzamide    (compound 88),-   5-(bis(2-bromoethyl)amino)-4-cyano-N-(3-(dimethylamino)propyl)-N-methyl-2-nitrobenzamide    (compound 89),-   2-((2-bromoethyl)(5-(4-methylpiperazine-1-carbonyl)-2-(methylsulfonyl)-4-nitrophenyl)amino)ethyl    methanesulfonate (compound 310),-   2-((2-bromoethyl)(5-(4-ethylpiperazine-1-carbonyl)-2-(methylsulfonyl)-4-nitrophenyl)amino)ethyl    methanesulfonate (compound 311),-   2-((2-bromoethyl)(5-(4-isopropylpiperazine-1-carbonyl)-2-(methylsulfonyl)-4-nitrophenyl)amino)ethyl    methanesulfonate (compound 312),-   2-((2-bromoethyl)(2-(ethylsulfonyl)-5-(4-methylpiperazine-1-carbonyl)-4-nitrophenyl)amino)ethyl    methanesulfonate (compound 313),-   2-((2-bromoethyl)(5-(4-ethylpiperazine-1-carbonyl)-2-(ethylsulfonyl)-4-nitrophenyl)amino)ethyl    methanesulfonate (compound 314),-   2-((2-bromoethyl)(2-(ethylsulfonyl)-5-(4-isopropylpiperazine-1-carbonyl)-4-nitrophenyl)amino)ethyl    methanesulfonate (compound 315),-   2-((2-bromoethyl)(2-(methylsulfonyl)-5-((2-morpholinoethyl)carbamoyl)-4-nitrophenyl)amino)ethyl    methanesulfonate (compound 316),-   2-((2-bromoethyl)(5-(methyl(2-morpholinoethyl)carbamoyl)-2-(methylsulfonyl)-4-nitrophenyl)amino)ethyl    methanesulfonate (compound 317),-   2-((2-bromoethyl)(2-(methylsulfonyl)-5-((3-morpholinopropyl)carbamoyl)-4-nitrophenyl)amino)ethyl    methanesulfonate (compound 318),-   2-((2-bromoethyl)(5-(methyl(3-morpholinopropyl)carbamoyl)-2-(methylsulfonyl)-4-nitrophenyl)amino)ethyl    methanesulfonate (compound 319),-   2-((2-bromoethyl)(5-((2-(4-methylpiperazin-1-yl)ethyl)carbamoyl)-2-(methylsulfonyl)-4-nitrophenyl)amino)ethyl    methanesulfonate (compound 320),-   2-((2-bromoethyl)(5-(methyl(2-(4-methylpiperazin-1-yl)ethyl)carbamoyl)-2-(methylsulfonyl)-4-nitrophenyl)amino)ethyl    methanesulfonate (compound 321),-   2-((2-bromoethyl)(5-((3-(4-methylpiperazin-1-yl)propyl)carbamoyl)-2-(methylsulfonyl)-4-nitrophenyl)amino)ethyl    methanesulfonate (compound 322),-   2-((2-bromoethyl)(5-(methyl(3-(4-methylpiperazin-1-yl)propyl)carbamoyl)-2-(methylsulfonyl)-4-nitrophenyl)amino)ethyl    methanesulfonate (compound 323),-   2-((2-bromoethyl)(5-((2-(dimethylamino)ethyl)carbamoyl)-2-(methylsulfonyl)-4-nitrophenyl)amino)ethyl    methanesulfonate (compound 324),-   2-((2-bromoethyl)(5-((2-(dimethylamino)ethyl)(methyl)carbamoyl)-2-(methylsulfonyl)-4-nitrophenyl)amino)ethyl    methanesulfonate (compound 325),-   2-((2-bromoethyl)(5-((3-(dimethylamino)propyl)carbamoyl)-2-(methylsulfonyl)-4-nitrophenyl)amino)ethyl    methanesulfonate (compound 326),-   2-((2-bromoethyl)(5-((3-(dimethylamino)propyl)(methyl)carbamoyl)-2-(methylsulfonyl)-4-nitrophenyl)amino)ethyl    methanesulfonate (compound 327),-   2-((2-bromoethyl)(2-(ethylsulfonyl)-5-((2-morpholinoethyl)carbamoyl)-4-nitrophenyl)amino)ethyl    methanesulfonate (compound 328),-   2-((2-bromoethyl)(2-(ethylsulfonyl)-5-(methyl(2-morpholinoethyl)carbamoyl)-4-nitrophenyl)amino)ethyl    methanesulfonate (compound 329),-   2-((2-bromoethyl)(2-(ethylsulfonyl)-5-((3-morpholinopropyl)carbamoyl)-4-nitrophenyl)amino)ethyl    methanesulfonate (compound 330),-   2-((2-bromoethyl)(2-(ethylsulfonyl)-5-(methyl(3-morpholinopropyl)carbamoyl)-4-nitrophenyl)amino)ethyl    methanesulfonate (compound 331),-   2-((2-bromoethyl)(2-(ethylsulfonyl)-5-((2-(4-methylpiperazin-1-yl)ethyl)carbamoyl)-4-nitrophenyl)amino)ethyl    methanesulfonate (compound 332),-   2-((2-bromoethyl)(2-(ethylsulfonyl)-5-(methyl(2-(4-methylpiperazin-1-yl)ethyl)carbamoyl)-4-nitrophenyl)amino)ethyl    methanesulfonate (compound 333),-   2-((2-bromoethyl)(2-(ethylsulfonyl)-5-((3-(4-methylpiperazin-1-yl)propyl)carbamoyl)-4-nitrophenyl)amino)ethyl    methanesulfonate (compound 334),-   2-((2-bromoethyl)(2-(ethylsulfonyl)-5-(methyl(3-(4-methylpiperazin-1-yl)propyl)carbamoyl)-4-nitrophenyl)amino)ethyl    methanesulfonate (compound 335),-   2-((2-bromoethyl)(5-((2-(dimethylamino)ethyl)carbamoyl)-2-(ethylsulfonyl)-4-nitrophenyl)amino)ethyl    methanesulfonate (compound 336),-   2-((2-bromoethyl)(5-((2-(dimethylamino)ethyl)(methyl)carbamoyl)-2-(ethylsulfonyl)-4-nitrophenyl)amino)ethyl    methanesulfonate (compound 337),-   2-((2-bromoethyl)(5-((3-(dimethylamino)propyl)carbamoyl)-2-(ethylsulfonyl)-4-nitrophenyl)amino)ethyl    methanesulfonate (compound 338),-   2-((2-bromoethyl)(5-((3-(dimethylamino)propyl)(methyl)carbamoyl)-2-(ethylsulfonyl)-4-nitrophenyl)amino)ethyl    methanesulfonate (compound 339),-   3-(5-((2-bromoethyl)(2-((methylsulfonyl)oxy)ethyl)amino)-4-(methylsulfonyl)-2-nitrobenzamido)propanoic    acid (compound 340),-   4-(5-((2-bromoethyl)(2-((methylsulfonyl)oxy)ethyl)amino)-4-(methylsulfonyl)-2-nitrobenzamido)butanoic    acid (compound 341),-   3-(5-((2-bromoethyl)(2-((methylsulfonyl)oxy)ethyl)amino)-N-methyl-4-(methylsulfonyl)-2-nitrobenzamido)propanoic    acid (compound 342),-   4-(5-((2-bromoethyl)(2-((methylsulfonyl)oxy)ethyl)amino)-N-methyl-4-(methylsulfonyl)-2-nitrobenzamido)butanoic    acid (compound 343),-   3-(5-((2-bromoethyl)(2-((methylsulfonyl)oxy)ethyl)amino)-4-(ethylsulfonyl)-2-nitrobenzamido)propanoic    acid (compound 344),-   4-(5-((2-bromoethyl)(2-((methylsulfonyl)oxy)ethyl)amino)-4-(ethylsulfonyl)-2-nitrobenzamido)butanoic    acid (compound 345),-   3-(5-((2-bromoethyl)(2-((methylsulfonyl)oxy)ethyl)amino)-4-(ethylsulfonyl)-N-methyl-2-nitrobenzamido)propanoic    acid (compound 346),-   4-(5-((2-bromoethyl)(2-((methylsulfonyl)oxy)ethyl)amino)-4-(ethylsulfonyl)-N-methyl-2-nitrobenzamido)butanoic    acid (compound 347),-   2-((2-bromoethyl)(2-cyano-5-(4-methylpiperazine-1-carbonyl)-4-nitrophenyl)amino)ethyl    methanesulfonate (compound 352),-   2-((2-bromoethyl)(2-cyano-5-(4-ethylpiperazine-1-carbonyl)-4-nitrophenyl)amino)ethyl    methanesulfonate (compound 353),-   2-((2-bromoethyl)(2-cyano-5-(4-isopropylpiperazine-1-carbonyl)-4-nitrophenyl)amino)ethyl    methanesulfonate (compound 354),-   3-(5-((2-bromoethyl)(2-((methylsulfonyl)oxy)ethyl)amino)-4-cyano-2-nitrobenzamido)propanoic    acid (compound 355),-   4-(5-((2-bromoethyl)(2-((methylsulfonyl)oxy)ethyl)amino)-4-cyano-2-nitrobenzamido)butanoic    acid (compound 356),-   3-(5-((2-bromoethyl)(2-((methylsulfonyl)oxy)ethyl)amino)-4-cyano-N-methyl-2-nitrobenzamido)propanoic    acid (compound 357),-   4-(5-((2-bromoethyl)(2-((methylsulfonyl)oxy)ethyl)amino)-4-cyano-N-methyl-2-nitrobenzamido)butanoic    acid (compound 358),-   2-((2-bromoethyl)(2-cyano-5-((2-morpholinoethyl)carbamoyl)-4-nitrophenyl)amino)ethyl    methanesulfonate (compound 359),-   2-((2-bromoethyl)(2-cyano-5-(methyl(2-morpholinoethyl)carbamoyl)-4-nitrophenyl)amino)ethyl    methanesulfonate (compound 360),-   2-((2-bromoethyl)(2-cyano-5-((3-morpholinopropyl)carbamoyl)-4-nitrophenyl)amino)ethyl    methanesulfonate (compound 361),-   2-((2-bromoethyl)(2-cyano-5-(methyl(3-morpholinopropyl)carbamoyl)-4-nitrophenyl)amino)ethyl    methanesulfonate (compound 362),-   2-((2-bromoethyl)(2-cyano-5-((2-(4-methylpiperazin-1-yl)ethyl)carbamoyl)-4-nitrophenyl)amino)ethyl    methanesulfonate (compound 363),-   2-((2-bromoethyl)(2-cyano-5-(methyl(2-(4-methylpiperazin-1-yl)ethyl)carbamoyl)-4-nitrophenyl)amino)ethyl    methanesulfonate (compound 364),-   2-((2-bromoethyl)(2-cyano-5-((3-(4-methylpiperazin-1-yl)propyl)carbamoyl)-4-nitrophenyl)amino)ethyl    methanesulfonate (compound 365),-   2-((2-bromoethyl)(2-cyano-5-(methyl(3-(4-methylpiperazin-1-yl)propyl)carbamoyl)-4-nitrophenyl)amino)ethyl    methanesulfonate (compound 366),-   2-((2-bromoethyl)(2-cyano-5-((2-(dimethylamino)ethyl)carbamoyl)-4-nitrophenyl)amino)ethyl    methanesulfonate (compound 367),-   2-((2-bromoethyl)(2-cyano-5-((2-(dimethylamino)ethyl)(methyl)carbamoyl)-4-nitrophenyl)amino)ethyl    methanesulfonate (compound 368),-   2-((2-bromoethyl)(2-cyano-5-((3-(dimethylamino)propyl)carbamoyl)-4-nitrophenyl)amino)ethyl    methanesulfonate (compound 369), and-   2-((2-bromoethyl)(2-cyano-5-((3-(dimethylamino)propyl)(methyl)carbamoyl)-4-nitrophenyl)amino)ethyl    methanesulfonate (compound 370).

In a second aspect of the invention there is provided a compound ofFormula (II)

wherein W represents Cl, Br, I, OSO₂R,

X represents Cl, Br, I, OSO₂R,

each R independently represents a lower C₁₋₆ alkyl group,

Z is selected from any of the radicals of Formula (IIa)

-   -   where        -   R₁ represents H, or a lower C₁₋₆ alkyl group,        -   n represents 2 to 6        -   * represents a point of attachment to Formula II

or a pharmaceutically acceptable salt thereof.

In a particular embodiment of the second aspect, the invention providesa compound of Formula (IIb)

wherein R represents a lower C₁₋₆ alkyl group,

Z is selected from any of the radicals of Formula (IIc)

-   -   where        -   n represents 2 to 6        -   * represents a point of attachment to Formula llb

or a pharmaceutically acceptable salt thereof.

In one embodiment the compound of Formula (II) comprises a compoundrepresented by formula (IId),

wherein n represents 2 to 6,

W represents Cl, Br, I, OSO₂R,

X represents Cl, Br, I, OSO₂R,

R represents a lower C₁₋₆ alkyl group,

R₁ represents H, or a lower C₁₋₆ alkyl group.

In another embodiment W is bromine or iodine.

In another embodiment X is bromine or OSO₂Me.

In another embodiment R₁ is hydrogen.

In another embodiment n represents 2 or 3

In one embodiment the compound of Formula (II) is selected from acompound represented by formula (IIe),

wherein n represents 2 to 6.

In another embodiment the compound of Formula (II) is selected from:

-   2-((2-bromoethyl)(4-nitro-2-((2-(phosphonooxy)ethyl)carbamoyl)phenyl)amino)ethyl    methanesulfonate (compound 60),-   2-((2-bromoethyl)(4-nitro-2-((3-(phosphonooxy)propyl)carbamoyl)phenyl)amino)ethyl    methanesulfonate (compound 61),

In another embodiment the compound of Formula (II) is selected from acompound represented by formula (IIf),

wherein n represents 2 to 6,

W represents Cl, Br, I, OSO₂R,

X represents Cl, Br, I, OSO₂R,

each R independently represents a lower C₁₋₆ alkyl group,

R₁ represents H, or a lower C₁₋₆ alkyl group.

In another embodiment W is bromine or iodine.

In another embodiment X is bromine or OSO₂Me.

In another embodiment R₁ is hydrogen.

In another embodiment n represents 2 or 3

In another embodiment the compound of Formula (II) is selected from acompound represented by formula (IIg),

wherein n represents 2 to 6,

In another embodiment n represents 2 or 3

In another embodiment the compound of Formula (II) as claimed isselected from:

-   2-((2-bromoethyl)(2-((2-hydroxyethyl)carbamoyl)-4-nitrophenyl)amino)ethyl    methanesulfonate (compound 64),-   2-((2-bromoethyl)(2-((3-hydroxypropyl)carbamoyl)-4-nitrophenyl)amino)ethyl    methanesulfonate (compound 66),

In a particular embodiment, the solubility of the compound of the firstor second aspect is greater than about 95 mM when determined inPhosphate Buffered Saline (PBS) containing 2 equivalents of sodiumbicarbonate.

In a particular embodiment, the solubility of the compound of the firstor second aspect is greater than about 10 mM when determined in LactateBuffer at pH=4.

In a third aspect there is provided a method of cell ablation comprisingthe use of a compound of the first or second aspect or a mixturethereof. In a particular embodiment, the compound is a prodrug capableof activation by contact with a) at least one nitroreductase enzyme,and/or b) a low oxygen (hypoxic) environment.

In a particular embodiment, the method of cell ablation comprises:

-   -   a. selecting a compound of formula (I) which is substantially        resistant to AKR1C3 enzyme metabolism;    -   b. contacting the compound of step a. with        -   i. at least one nitroreductase enzyme, and/or        -   ii. a hypoxic environment, to produce a cytotoxic metabolite            capable of ablating the cell;    -   c. contacting the cell with the cytotoxic metabolite;

wherein the compound of formula (I) is as defined in the first aspect.

In a particular embodiment, the method of cell ablation comprises:

-   -   a. selecting a compound of formula (II) which is substantially        resistant to AKR1C3 enzyme metabolism;    -   b. contacting the compound of step a. with        -   i. at least one nitroreductase enzyme, and/or        -   ii. a hypoxic environment, to produce a cytotoxic metabolite            capable of ablating the cell;        -   c. contacting the cell with the cytotoxic metabolite;

wherein the compound of formula (II) is as defined in the second aspect.

Preferably, the hypoxic environment is a hypoxic region of a tumour.

Preferably the prodrug is capable of providing a substantial bystandereffect which results in cell ablation.

In one embodiment of the third aspect, the cell is a tumour cell intissue in a subject.

In one embodiment of the third aspect the cell is a mammalian cell.

In one embodiment of the third aspect, the method of cell ablation is amethod of cancer treatment comprising the administration of the compoundto a subject. Preferably, the amount of compound administered is atherapeutically effective amount. Preferably, this amount is betweenabout 20% to 100% of the maximum tolerated dose of said subject.

In a particular embodiment of the third aspect the compound isadministered to a subject in combination with at least onenitroreductase enzyme capable of metabolising the compound. In aparticular embodiment, the compound is administered to a subject incombination with a therapy that results in expression of an exogenousnitroreductase enzyme within, or therapeutically proximate to, a tumour.In a further embodiment, the at least one nitroreductase enzyme isencoded for by the nfsB and/or the nfsA gene of either E. coli or byorthologous genes in other bacterial species. In a particularembodiment, the nitroreductase is a nitroreductase described inWO/2012/008860, which includes mutant nitroreductases and functionallyequivalent variants of the nitroreductase described therein.

In a particular embodiment of the third aspect, the method includes thestep of irradiating the cell. Preferably, irradiation is carried outbefore, concurrently with, or after administration of the prodrug.Preferably, the amount of absorbed radiation is 15 gray (Gy).

In a particular embodiment of the third aspect, the method includes theadministration of a compound of the first or second aspect or mixturethereof in conjunction with GDEPT (gene-directed enzyme prodrugtherapy), VDEPT (virus-directed enzyme prodrug therapy), CDEPT(clostridia-directed enzyme prodrug therapy) or ADEPT (antibody-directedenzyme prodrug therapy).

In a particular embodiment, the CDEPT comprises use of a Clostridiamicroorganism that is selective for colonising the necrosis found intumours. Preferably, the Clostridia microorganism is a recombinantmicroorganism comprising one or more genes expressing a nitroreductaseexogenous to the Clostridia microorganism. Preferably, thenitroreductase enzyme is encoded for by the nfsB and/or the nfsA gene ofeither E. coli or by orthologous genes in other bacterial species

In a particular embodiment of the third aspect the method furtherincludes the dissolution of the compound in an aqueous solution.

In a fourth aspect, the invention provides the use of a compound of thefirst or second aspect or a mixture thereof in the manufacture of acomposition to ablate a cell. In a particular embodiment, the compoundis a prodrug capable of activation by contact with a) at least onenitroreductase enzyme, and/or b) a low oxygen (hypoxic) environment.

In a fifth aspect, the invention provides the use of a compound asdefined in the first or second aspect in the manufacture of acomposition for the treatment of cancer or a hyperproliferativecondition.

In a sixth aspect, the invention provides the use of a compound asdefined in the first or second aspect for the treatment of cancer or ahyperproliferative condition.

In a seventh aspect of the invention there is provided a method oftreatment of cancer or a hyperproliferative condition wherein a compoundof the first or second aspect or a mixture thereof is administered in atherapeutically effective amount to a tumour cell, or therapeuticallyproximate to a tumour cell, in a subject.

In a particular embodiment of the seventh aspect, the therapeuticallyeffective amount administered is between about 20% to 100% of themaximum tolerated dose of said subject.

In a particular embodiment of the seventh aspect the compound of thefirst or second aspect or mixture thereof is administered in conjunctionwith at least one nitroreductase enzyme. In a particular embodiment, thecompound is administered to a subject in combination with a therapy thatresults in expression of an exogenous nitroreductase enzyme within, ortherapeutically proximate to, a tumour.

In a particular embodiment of the seventh aspect, the at least onenitroreductase enzyme is encoded for by the nfsA or nfsB gene of eitherE. coli or by orthologous genes in other bacterial species. In aparticular embodiment, the nitroreductase is a nitroreductase describedin WO/2012/008860, which includes mutant nitroreductases andfunctionally equivalent variants of the nitroreductase describedtherein.

In a particular embodiment the method further comprises the activationof the compound of the first or second aspect by contact with thenitroreductase enzyme.

In a particular embodiment of the seventh aspect, the compound of thefirst or second aspect or mixture thereof is administered in conjunctionwith GDEPT (gene-directed enzyme prodrug therapy), VDEPT (virus-directedenzyme prodrug therapy), CDEPT (clostridia-directed enzyme prodrugtherapy) or ADEPT (antibody-directed enzyme prodrug therapy).

In a particular embodiment, the CDEPT comprises use of a Clostridiamicroorganism that is selective for colonising the necrosis found intumours. Preferably, the Clostridia microorganism is a recombinantmicroorganism comprising one or more genes expressing a nitroreductaseexogenous to the Clostridia microorganism.

In a particular embodiment of the seventh aspect, the method of cancertreatment further includes the step of irradiating the tumour cells.Preferably, the irradiation is carried out before, concurrently with, orafter the administration of the compound. Preferably, the amount ofabsorbed radiation is 15 gray (Gy).

In a particular embodiment of the seventh aspect, the method furtherincludes the dissolution of the compound in an aqueous solution.

In an eighth aspect there is provided a pharmaceutical compositioncomprising a therapeutically effective amount of a compound of the firstor second aspect, or a mixture thereof, and a pharmaceuticallyacceptable excipient, adjuvant, carrier, buffer or stabiliser.

In a particular embodiment, the composition is soluble in aqueoussolution. Preferably, the solubility of the compound of the first orsecond aspect as found in the composition is greater than 95 mM whendetermined in Phosphate Buffered Saline (PBS) containing 2 equivalentsof sodium bicarbonate.

In a ninth aspect, the invention provides a method of determiningsensitivity of prodrugs to metabolism by AKR1C3 enzymes. The inventorshave shown that the method of the ninth aspect provides a high celldensity MCL screen that is surprisingly effective in detecting ‘falsenegatives’ from a two dimensional in vitro IC50 screen. The method alsohas particular utility in identification of bona fide AKR1C3-negativeprodrugs, such as those of the present invention.

Further aspects of the invention, which should be considered in all itsnovel aspects, will become apparent to those skilled in the art uponreading of the following description which provides at least one exampleof a practical application of the invention.

BRIEF DESCRIPTION OF DRAWINGS

Embodiments of the invention will now be described, by way of exampleonly, with reference to the accompanying drawings in which:

FIG. 1 shows representative phosphates of dibromo mustards of Formula I.

FIG. 2 shows representative dibromo mustard alcohols of Formula I.

FIG. 3A shows representative dibromo mustards bearing substitutedpiperazine carboxamide sidechains of Formula I.

FIG. 3B shows representative bromomesylate mustards bearing substitutedpiperazine carboxamide sidechains of Formula I.

FIG. 4 shows representative 4-methylsulfonyl dibromo mustards bearingamine sidechains of Formula I.

FIG. 5A shows representative 4-ethylsulfonyl dibromo mustards bearingamine sidechains of Formula I.

FIG. 5B shows representative 4-methylsulfonyl bromomesylate mustardsbearing amine sidechains of Formula I.

FIG. 5C shows representative 4-ethylsulfonyl bromomesylate mustardsbearing amine sidechains of Formula I.

FIG. 6A shows representative dibromo mustards bearing acid sidechains ofFormula I.

FIG. 6B shows representative bromomesylate mustards bearing acidsidechains of Formula I.

FIG. 7A shows representative phosphates of bromomesylate mustards ofFormula II.

FIG. 7B shows representative bromomesylate mustard alcohols of FormulaII.

FIG. 7C shows representative 4-cyano dibromo mustard alcohols,phosphates and amine sidechain bearing compounds of Formula I.

FIG. 7D shows representative 4-cyano bromomesylate mustard alcohols,phosphates and amine sidechain bearing compounds of Formula I.

FIG. 7E shows representative 4-cyano dibromo mustards bearing acid andamine sidechains of Formula I.

FIG. 7F shows representative 4-cyano bromomesylate mustards bearing acidand amine sidechains of Formula I.

Compounds X, XI and XII in FIG. 8, compound XII in FIG. 9A, andcompounds XI and XXIII in FIG. 9B each comprise three R groups. The twoR groups attached to the —OSO₂ substituents will be identical usingthese methods of synthesis. The other R group attached to the sulphone—SO₂ group can vary independently of the other R groups while stillbeing within the range defined for R in formula (I) where R represents alower C₁₋₆ alkyl group.

FIG. 8 shows a general synthetic scheme for the synthesis of4-alkylsulfone prodrugs of Formula I.

FIG. 9A shows a preferred general synthetic scheme for the synthesis of4-alkylsulfone prodrugs bearing a 1-position tertiary carboxamide ofFormula I.

FIG. 9B shows a general synthetic scheme for the synthesis of4-alkylsulfone prodrugs bearing acid sidechains of Formula I.

FIG. 9C shows a general synthetic scheme for synthesis of 4-cyanoprodrugs of Formula I.

FIG. 9D shows a preferred general synthetic scheme for the synthesis of4-cyano prodrugs bearing a 1-position tertiary carboxamide of Formula I.

FIG. 9E shows a general synthetic scheme for the synthesis of 4-cyanoprodrugs bearing acid sidechains of Formula I.

FIG. 9F shows a scheme for synthesis of alcohol compound 14 (FIG. 2).

FIG. 9G shows a scheme for synthesis of alcohol compound 18 (FIG. 2).

FIG. 9H shows a scheme for synthesis of alcohol compound 301 (FIG. 2).

FIG. 10 shows a scheme for synthesis of phosphates 10, and 11 and 300(FIG. 1).

FIG. 11 shows a scheme for synthesis of prodrugs 22 to 27 (FIG. 3A).

FIG. 12 shows a general scheme for synthesis of prodrugs of Formula II.

FIG. 13 shows a scheme for synthesis of alcohol 64 and phosphate 60.

FIG. 14 shows the aqueous solubility of prior art prodrug 4 (Scheme 1)versus prodrugs 10, 11, 23 and 300 of the present invention. TableFootnote: ^(a)Determined in α-Minimal Essential Media (α-MEM) containing5% Fetal Calf Serum (FCS). ^(b)Determined in Phosphate Buffered Saline(PBS) containing 2 equivalents of sodium bicarbonate. ^(c)Determined inLactate Buffer at pH=4.

FIG. 15 shows a comparison of IC50 (uM) of prodrugs of the prior art(PR-104A, 5 to 9) in HCT116 wild type cancer cells versus HCT116 cellsengineered to over-express the human two-electron reductasealdo-ketoreductase 1C3 (AKR1C3). Progressing across the series from 5 to9 is consistent with a relative loss of AKR1C3 metabolism inducedcytotoxicity compared to PR-104 in this low cell density assay, suchthat prodrug 9 appears to be AKR1C3-negative.

FIG. 16 shows a recombinant AKR1C3 metabolism assay for rate of loss ofNADPH co-factor. All of the prodrugs 5 to 9 are better substrates forAKR1C3 than PR-104A.

FIG. 17A shows clonogenic cell kill of PR-104A, prodrug 7 and prodrug 9in HCT116 wild type Multicellular Layers (MCLs) versus HCT116 MCLs wherethe cells are engineered to over-express the human two-electronreductase aldo-ketoreductase 1C3 (AKR1C3). Prodrugs 7 and 9 are bothcapable of producing AKR1C3-dependent cell ablation, when cells aregrown in three dimensional structures. Cytotoxic metabolites leaving thecell where they are produced are able to kill neighbouring cells. In alow cell density assay they are diluted into the assay media, protectingthe cell of production from cytotoxicity, essentially providing anAKR1C3 false negative (see FIG. 15). The inventors have found that MCLassays are essential for identifying prodrugs that are free of AKR1C3metabolism related cytotoxicity.

FIG. 17B shows the metabolism of compound 14 by members of the 55candidate nitroreductase over-expression library as measured by GFP SOSassay. The data presented is the fold increase of GFP SOS response(normalised to culture density) exhibited by microplate cultures of E.coli strain SOS-R4 over-expressing candidate nitroreductases whenchallenged for 6 h with 20 μM of compound 14, compared to anunchallenged control. Data are the average of 2 independent assays andthe error bars indicate ±1 standard deviation. The dashed line indicatesthe baseline activity for the empty plasmid control, and the data setscorresponding to the NfsA and NfsB family members are as marked. Inset:Metabolism of compound 14 was demonstrated for a selection of single andpoly mutant variants of NfsA_Ec and NfsA_Bs. Data sets were generated inidentical fashion to those described above for the 55 candidatenitroreductase library.

FIG. 17C shows the metabolism of compound 18 by members of the 55candidate nitroreductase over-expression library as measured by GFP SOSassay. The data presented is the fold increase of GFP SOS response(normalised to culture density) exhibited by microplate cultures of E.coli strain SOS-R4 over-expressing candidate nitroreductases whenchallenged for 6 h with 20 μM of compound 18, compared to anunchallenged control. Data are the average of 2 independent assays andthe error bars indicate ±1 standard deviation. The dashed line indicatesthe baseline activity for the empty plasmid control, and the data setscorresponding to the NfsA and NfsB family members are as marked. Inset:Metabolism of compound 18 was demonstrated for a selection of single andpoly mutant variants of NfsA_Ec and NfsA_Bs. Data sets were generated inidentical fashion to those described above for the 55 candidatenitroreductase library.

FIG. 17D shows the metabolism of compound 22 by members of the 55candidate nitroreductase over-expression library as measured by GFP SOSassay. The data presented is the fold increase of GFP SOS response(normalised to culture density) exhibited by microplate cultures of E.coli strain SOS-R4 over-expressing candidate nitroreductases whenchallenged for 6 h with 50 μM of compound 22, compared to anunchallenged control. Data are the average of 2 independent assays andthe error bars indicate ±1 standard deviation. The dashed line indicatesthe baseline activity for the empty plasmid control, and the data setscorresponding to the NfsA and NfsB family members are as marked. Inset:Metabolism of compound 22 was demonstrated for a selection of single andpoly mutant variants of NfsA_Ec and NfsA_Bs. Data sets were generated inidentical fashion to those described above for the 55 candidatenitroreductase library.

FIG. 17E shows the metabolism of compound 23 by members of the 55candidate nitroreductase over-expression library as measured by GFP SOSassay. The data presented is the fold increase of GFP SOS response(normalised to culture density) exhibited by microplate cultures of E.coli strain SOS-R4 over-expressing candidate nitroreductases whenchallenged for 6 h with 60 μM of compound 23, compared to anunchallenged control. Data are the average of 2 independent assays andthe error bars indicate ±1 standard deviation. The dashed line indicatesthe baseline activity for the empty plasmid control, and the data setscorresponding to the NfsA and NfsB family members are as marked. Inset:Metabolism of compound 23 was demonstrated for a selection of single andpoly mutant variants of NfsA_Ec and NfsA_Bs. Data sets were generated inidentical fashion to those described above for the 55 candidatenitroreductase library.

FIG. 17F shows the metabolism of compound 24 by members of the 55candidate nitroreductase over-expression library as measured by GFP SOSassay. The data presented is the fold increase of GFP SOS response(normalised to culture density) exhibited by microplate cultures of E.coli strain SOS-R4 over-expressing candidate nitroreductases whenchallenged for 6 h with 60 μM of compound 24, compared to anunchallenged control. Data are the average of 2 independent assays andthe error bars indicate ±1 standard deviation. The dashed line indicatesthe baseline activity for the empty plasmid control, and the data setscorresponding to the NfsA and NfsB family members are as marked. Inset:Metabolism of compound 24 was demonstrated for a selection of single andpoly mutant variants of NfsA_Ec and NfsA_Bs. Data sets were generated inidentical fashion to those described above for the 55 candidatenitroreductase library.

FIG. 17G shows the metabolism of compound 25 by members of the 55candidate nitroreductase over-expression library as measured by GFP SOSassay. The data presented is the fold increase of GFP SOS response(normalised to culture density) exhibited by microplate cultures of E.coli strain SOS-R4 over-expressing candidate nitroreductases whenchallenged for 6 h with 60 μM of compound 25, compared to anunchallenged control. Data are the average of 2 independent assays andthe error bars indicate ±1 standard deviation. The dashed line indicatesthe baseline activity for the empty plasmid control, and the data setscorresponding to the NfsA and NfsB family members are as marked. Inset:Metabolism of compound 25 was demonstrated for a selection of single andpoly mutant variants of NfsA_Ec and NfsA_Bs. Data sets were generated inidentical fashion to those described above for the 55 candidatenitroreductase library.

FIG. 17H shows the metabolism of compound 26 by members of the 55candidate nitroreductase over-expression library as measured by GFP SOSassay. The data presented is the fold increase of GFP SOS response(normalised to culture density) exhibited by microplate cultures of E.coli strain SOS-R4 over-expressing candidate nitroreductases whenchallenged for 6 h with 60 μM of compound 26, compared to anunchallenged control. Data are the average of 2 independent assays andthe error bars indicate ±1 standard deviation. The dashed line indicatesthe baseline activity for the empty plasmid control, and the data setscorresponding to the NfsA and NfsB family members are as marked. Inset:Metabolism of compound 26 was demonstrated for a selection of single andpoly mutant variants of NfsA_Ec and NfsA_Bs. Data sets were generated inidentical fashion to those described above for the 55 candidatenitroreductase library.

FIG. 17I shows the metabolism of compound 27 by members of the 55candidate nitroreductase over-expression library as measured by GFP SOSassay. The data presented is the fold increase of GFP SOS response(normalised to culture density) exhibited by microplate cultures of E.coli strain SOS-R4 over-expressing candidate nitroreductases whenchallenged for 6 h with 60 μM of compound 27, compared to anunchallenged control. Data are the average of 2 independent assays andthe error bars indicate ±1 standard deviation. The dashed line indicatesthe baseline activity for the empty plasmid control, and the data setscorresponding to the NfsA and NfsB family members are as marked. Inset:Metabolism of compound 27 was demonstrated for a selection of single andpoly mutant variants of NfsA_Ec and NfsA_Bs. Data sets were generated inidentical fashion to those described above for the 55 candidatenitroreductase library.

FIG. 17J shows the metabolism of compound 64 by members of the 55candidate nitroreductase over-expression library as measured by GFP SOSassay. The data presented is the fold increase of GFP SOS response(normalised to culture density) exhibited by microplate cultures of E.coli strain SOS-R4 over-expressing candidate nitroreductases whenchallenged for 6 h with 10 μM of compound 64, compared to anunchallenged control. Data are the average of 2 independent assays andthe error bars indicate ±1 standard deviation. The dashed line indicatesthe baseline activity for the empty plasmid control, and the data setscorresponding to the NfsA and NfsB family members are as marked. Inset:Metabolism of compound 64 was demonstrated for a selection of single andpoly mutant variants of NfsA_Ec and NfsA_Bs. Data sets were generated inidentical fashion to those described above for the 55 candidatenitroreductase library.

FIG. 17K shows purified enzyme kinetic data with compounds 14, 18, 22,23, 24, 25, 26, 27 and 64 for NfsA_Ec. Reactions contained 10 mM Tris-Cl(pH 7.0), 4% DMSO, 0.20 mM NADPH and varying compound concentrations.Reactions were initiated by addition of enzyme and changes in absorbancewere measured for 20 s at 400 nm on a spectrophotometer to monitorNTR-catalysed compound reduction. Non-linear regression analysis andMichaelis-Menten curve fitting was performed using Sigmaplot 10.0(Systat Software Inc.).

FIG. 17L shows UV/Vis spectroscopy measurements of the relative rates ofNfsB_Ec catalysed reduction for each test compound at 400 nm. Compounds14, 18, 22, 23, 24, 25, 26, 27 and 64 (600 μM) were added to NADPH (200μM) in 10 mM Tris-Cl pH 7.0. Reactions were initiated by enzyme addition(between 0.25 and 5 μg per reaction, determined empirically in pilotexperiments). Rates represent μmol of compound reduced per mg enzymeadded per minute. Data are the average of at least 4 independentmeasurements and the error bars indicate ±1 standard deviation.

FIG. 18A shows IC50 (uM) of prodrugs PR-104A and 14, 18, 22, 23, 24, 25,26, 27, 64 of the present invention, in HCT116 wild type cancer cellsversus HCT116 cells engineered to over-express the human two-electronreductase aldo-ketoreductase 1C3 (AKR1C3). All appear less susceptibleto AKR1C3-mediated cytotoxicity than PR-104A.

FIG. 18B shows IC50 (uM) of prodrugs PR-104A and 22, 23, 24, 25, 26, 27of the present invention, in H1299 wild type cancer cells versus H1299cells engineered to over-express the human two-electron reductasealdo-ketoreductase 1C3 (AKR1C3). All appear less susceptible toAKR1C3-mediated cytotoxicity than PR-104A.

FIG. 19A shows clonogenic cell kill of PR-104A compared to prodrugs 14,18, 22, 23, 24, 25, 26, 27, 64 of the present invention in HCT116 wildtype Multicellular Layers (MCLs) versus HCT116 MCLs where the cells areengineered to over-express the human two-electron reductasealdo-ketoreductase 1C3 (AKR1C3). Prodrugs 14, 18, 22, 23, 24, 25, 26, 27and 64 do not cause AKR1C3-dependant cytotoxicity.

FIG. 19B shows the calculated lipophilicity of compounds of formula ifcompared to their status with respect to demonstrating AKR1C3-dependentcytotoxicity. Footnotes for figure: ^(a)Determined using a trained ACDLabs (version 8) log P calculator. ^(b)AKR1C3-dependent metabolismstatus as determined by assessing clonogenic cell kill of the prodrugsin HCT116 wild type Multicellular Layers (MCLs) versus HCT116 MCLs wherethe cells are engineered to over-express the human two-electronreductase aldo-ketoreductase 1C3 (AKR1C3).

FIG. 20A shows IC50 (uM) of prodrugs PR-104A and 14, 18, 22, 23, 24, 25,26, 27, 64 of the present invention, in HCT116 wild type cancer cellsversus HCT116 cells engineered to over-express the bacterialtwo-electron reductase E coli NfsA. All prodrugs produce E coliNfsA-mediated cytotoxicity.

FIG. 20B shows IC50 (uM) of prodrugs PR-104A and 14, 18, 22, 23, 24, 25,26, 27, 64 of the present invention, in H1299 wild type cancer cellsversus H1299 cells engineered to over-express the bacterial two-electronreductase E coli NfsA. All prodrugs produce E coli NfsA-mediatedcytotoxicity.

FIG. 21 shows clonogenic cell kill of PR-104A compared to prodrugs 14,18, 22, 23, 24, 25, 26, 27, 64 of the present invention in HCT116 wildtype Multicellular Layers (MCLs) versus HCT116 MCLs seeded with 3% ofthe cells engineered to over-express the bacterial two-electronreductase E coli NfsA to assess bystander cell killing. All of theprodrugs 14, 18, 22, 23, 24, 25, 26, 27 and 64 display evidence ofmetabolism by the 3% E coli NfsA-expressing cells with bystander cellkilling of the 97% non-expressing neighbour cells.

FIG. 22A shows mean tumour volume (mm3) of 15% E coli NfsA-expressingHCT116 xenografts (containing 85% wild type cells) in NIH-III miceadministered a single dose of the prodrugs 10, 22 and 60 at doses of1000, 422 and 1330 umol/kg, respectively. All of the prodrugs displaysignificant E coli NfsA mediated anti-tumour efficacy. Mean tumourvolume of PR-104 at the human equivalent dose of 338 umol/kg is shown byway of reference.

FIG. 22B shows the median time to four times relative tumour volume(RTV4) and tumour growth delay (TGD) as a percentage of vehicle onlytreated tumour controls, for 15% E coli NfsA-expressing HCT116xenografts (containing 85% wild type cells) in NIH-III mice administereda single dose of the prodrugs PR-104, 10, 22 and 60 at doses of 338,1000, 422 and 1330 umol/kg, respectively.

FIG. 22C shows mean tumour volume (mm3) of 15% E coli NfsA-expressingH1299 xenografts (containing 85% wild type cells) in NIH-III miceadministered a single dose of the prodrugs 10, 22, 60 and 11 at doses of750, 422, 1330 and 1330 umol/kg, respectively. All of the prodrugsdisplay significant E coli NfsA mediated anti-tumour efficacy. Meantumour volume of PR-104 at the human equivalent dose of 225 mg/kg (388umol/kg) is shown by way of reference.

FIG. 22D shows the median time to four times relative tumour volume(RTV4) and tumour growth delay (TGD) as a percentage of vehicle onlytreated tumour controls, for 15% E coli NfsA-expressing H1299 xenografts(containing 85% wild type cells) in NIH-III mice administered a singledose of the prodrugs PR-104, 10, 22, 60 and 11 at doses of 338, 750,422, 1330 and 1330 umol/kg, respectively.

FIG. 22E shows mean tumour volume (mm3) of 15% E coli NfsA-expressingH1299 xenografts (containing 85% wild type cells) in NIH-III miceadministered a dose of the prodrugs 22, 23 and 26 at 500 umol/kg twicedaily (ie BID) for a total single daily dose of 1000 umol/kg. All of theprodrugs display significant E coli NfsA mediated anti-tumour efficacy.

FIG. 22F shows the median time to four times relative tumour volume(RTV4) and tumour growth delay (TGD) as a percentage of vehicle onlytreated tumour controls, for 15% E coli NfsA-expressing H1299 xenografts(containing 85% wild type cells) in NIH-III mice administered a singledose of the prodrugs 22, 23 and 26 at a dose of 1000 umol/kg (500umol/kg BID).

FIG. 23 shows oxic and anoxic IC50 (uM) of prodrugs 14, 22, 18 and 301in the HCT116, H460, H1299 and SiHa wild type cancer cell lines and theHCT116 POR cell line, an HCT116 cell line that has been engineered toover-express the human one-electron reductase Cytochrome P450 reductase.In each example exposure of the cells to anoxia leads to selectivemetabolism of the prodrugs producing metabolites of increasedcytotoxicity, resulting in Hypoxic Cytotoxicity Ratios (HCR) rangingfrom 11 to 28-fold for compounds 18 and 301 in the wild type cancer celllines. Larger HCRs were observed for compounds 14 and 22 in the HCT116POR cell line indicating increased prodrug metabolism and thereforecytotoxicity in cells over-expressing this human one-electron reductase.

FIG. 24 shows calculated log cell kill (LCK) for ‘prodrug only’ or ‘15Gyradiation+prodrug’ in HCT116 tumour xenografts engineered toover-express the human one-electron reductase Cytochrome P450 reductase.PR-104 (345 μmol/kg) and prodrug 300 (1330 μmol/kg) in mice receiving noradiation (black bars) or in mice which have received 15Gy radiation(grey bars). Prodrug 300 displays significant hypoxic cell kill in vivo.

FIG. 25 shows calculated log cell kill (LCK) versus vehicle onlycontrols for ‘prodrug only’ (single agent activity) or ‘10Gyradiation+prodrug’ in wild type SiHa tumour xenografts grownsubcutaneously in NIH-III nude mic. Mice were administered compound 22at 422 μmol/kg. LCK of compound 22 in mice receiving no radiation (blackbars) or in mice which have received both compound 22 (422 umol/kg) and10Gy radiation (light grey bars). LCK of radiation alone (mice receivevehicle only and not compound 22) is shown in light grey bars with blackstripes. Prodrug 22 displays significant hypoxic cell kill in vivo asdetermined by clonogenic cell kill of tumour cells that are notsterilised by 10Gy of radiation.

FIG. 26 shows calculated log cell kill (LCK) versus vehicle onlycontrols for ‘prodrug only’ (single agent activity) or ‘10Gyradiation+prodrug’ in wild type SiHa tumour xenografts grownsubcutaneously in NIH-III nude mice. Mice were administered compound300, 11 and 23 at 1330, 1330 and 1000 umol/kg, respectively. LCK of testcompounds in mice receiving no radiation (black bars) or in mice whichhave received both test compounds and 10Gy radiation (light grey bars).LCK of radiation alone (mice receive vehicle only and not testcompounds) is shown in light grey bars with black stripes. Prodrugs 300,11 and 23 display significant hypoxic cell kill in vivo as determined byclonogenic cell kill of tumour cells that are not sterilised by 10Gy ofradiation. LCK for ‘prodrug plus radiation’ was >4 (ie off-scale in thisassay) for 4/4 mice treated with compounds 300 and 23 (denoted by anupward point arrow). LCK for ‘prodrug plus radiation’ was >4 (ieoff-scale in this assay) for 1/4 mice treated with compound 11 (denotedby +).

DETAILED DESCRIPTION OF THE INVENTION

Definitions

PR-104 is a phosphate ester pre-prodrug of the alcohol PR-104A. PR-104is also called2-((2-bromoethyl)(2,4-dinitro-6-((2-(phosphonooxy)ethyl)carbamoyl)phenyl)amino)ethylmethanesulfonate.

PR-104A also called2-((2-bromoethyl)(2-((2-hydroxyethyl)carbamoyl)-4,6-dinitrophenyl)amino)ethylmethanesulfonate

“Nitroreductase”—an enzyme that catalyses the reduction of a nitrofunctional group (—NO₂) or quinone functional group. Nitroreductases asreferred to herein may be either endogenous or exogenous.

“Prodrug”—An inactive compound that is converted to a reactive cytotoxicmetabolite once activated. Preferably activation occurs withinnitroreductase expressing target cells within the local tumourmicroenvironment by reduction of a nitro group. Prodrugs may beactivated by reduction by a bacterial nitroreductase independent oftissue oxygen concentration or by reduction by a human nitroreductase intissues lacking oxygen (hypoxic tissues).

“Activation” or “metabolism” with reference to prodrugs refers to thecatalytic reduction process that a prodrug may undergo following contactwith an enzyme. The prodrug may be activated/metabolised to yieldalternative compounds such as cytotoxic metabolites that may havebeneficial activity for therapeutic applications.

“Ablation” is to be considered in its broadest context and as well asmeaning the complete ceasing of the function of the target beingablated, is also intended to encompass any degree of suppression of thefunction of the target where the target includes but is not limited to acell.

“Cell” refers to a biological sub-unit that is specialized in carryingout a particular function or functions. For the purposes of theinvention as defined herein, the term “cell” also encompasses the mediumin which the cell is found. For example this may mean a hypoxic regionof a tumour or the cell matrix which supports the cell in vivo or invitro.

“Endogenous”—Naturally occurring, originating or produced within anorganism, tissue, or cell. For example endogenous enzymes in a mammalare enzymes that are naturally present in mammalian cells.

“Exogenous”—Originating or produced outside of an organism, tissue, orcell. For example exogenous enzymes in a mammal are foreign enzymes thatdo not occur in mammalian cells. For example bacterial enzymes that mayhave been introduced through genetic manipulations.

“Hypoxic” as referred to herein refers to a concentration of oxygen intissue that is significantly lower the normal physiologicalconcentration of oxygen in healthy well perfused tissue, in particularoxygen tensions below approximately 1% (10,000 parts per million oxygen;7.6 mmHg).

“Bystander effect”—this effect is triggered by treatment of a targetcell with a cytotoxic prodrug metabolite and refers to the secondaryablation effect on cells or tissues in the local microenvironment to thetarget cell. Without wishing to be bound by theory, the bystander effectis believed to be caused by the diffusion of cytotoxic prodrugmetabolites (activated prodrugs) from the site of production to affectunmodified cells separate from the target cell.

“Treatment” is to be considered in its broadest context. The term doesnot necessarily imply that a subject is treated until total recovery.Accordingly, “treatment” broadly includes, for example, the prevention,amelioration or management of one or more symptoms of a disorder, theseverity of one or more symptoms and preventing or otherwise reducingthe risk of developing secondary complications.

“Prevention” of disease should not be taken to imply that diseasedevelopment is completely prevented, and includes delay of diseasedevelopment.

“Nitrobenzamide mustards” refers to any compound possessing a benzenering that is substituted with nitro, carboxamide and aniline mustardfunctionalities.

“Nitrobenzamide mustard alcohols” refers to any compound possessing abenzene ring that is substituted with nitro, carboxamide and anilinemustard functionalities where the carboxamide substituent furthercontains an alcohol moiety.

“AKR1C3 enzyme” refers to the human enzyme aldo-keto reductase 1C3. Thealdo-keto reductases (AKRs) are a superfamily of cytosolic enzymes thatare involved in the reduction of aldehydes and ketones to theircorresponding primary and secondary alcohols, respectively, from avariety of endogenous and exogenous substrates (Jez et al., 1997). AKRsrequire the presence of a cofactor NADPH in order to catalyze thereduction of carbonyl groups (Schlegel et al., 1998). The human AKRs areclassed into three families—AKR1, AKR6 and AKR7—of which AKR1 is thebest characterized in terms of structure and function (Penning andDrury, 2007). The AKR1C subfamily includes AKR1C1-4, with all fourenzymes having hydroxysteroid dehydrogenase (HSD) activity (Penning etal., 2000). The genes encoding AKR1C1-4 share more than 86% amino acidsequence identity, and show differences in substrate andregiospecificity of the sites metabolized (Penning and Byrns, 2009).AKR1C3 is the enzyme responsible for the reduction of Prostaglandin D2(PGD2) in humans.

“Substantially resistant to AKR1C3 enzyme metabolism” as referred toherein refers to a compound that exhibits a very low or substantiallyzero degree of metabolism by the human AKR1C3 enzyme when compared to acompound that is readily metabolised by human AKR1C3. AKR1C3 metabolismcan be demonstrated by incubating test compounds and NADPH co-factorwith recombinant AKR1C3 protein and assaying for the loss of NADPHco-factor, where a loss of co-factor indicates enzymatic metabolism ofthe compounds. Prodrugs of the present invention that are metabolised byAKR1C3 demonstrate increased clonogenic cell kill in multicellularlayers of cells engineered to over-express AKR1C3 relative tomulticellular layers of wild type isogenic cells, whereas compounds ofthe present invention that are substantially resistant to AKR1C3 enzymemetabolism demonstrate an inability to provide increased clonogenic cellkill in multicellular layers of cells engineered to over-express AKR1C3relative to multicellular layers of wild type isogenic cells.

“Pharmaceutically acceptable” means that which is useful in preparing apharmaceutical composition that is generally safe, non-toxic, andneither biologically nor otherwise undesirable and includes that whichis acceptable for veterinary as well as human pharmaceutical use.

“Pharmaceutically acceptable salts” of a compound means salts that arepharmaceutically acceptable, as defined herein, and that possess thedesired pharmacological activity of the parent compound.

Such salts include:

-   -   acid addition salts formed with inorganic acids such as        hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,        phosphoric acid and the like; or formed with organic acids such        as acetic acid, methanesulfonic acid, maleic acid, tartaric        acid, citric acid and the like; or    -   salts formed when an acidic proton present in the parent        compound either is replaced by a metal ion, e.g. an alkali metal        ion, an alkaline earth ion, or an aluminium ion; or coordinates        with an organic or inorganic base. Acceptable organic bases        include ethanolamine, diethanolamine, N-methylglucamine,        triethanolamine and the like. Acceptable inorganic bases include        aluminium hydroxide, calcium hydroxide, potassium hydroxide,        sodium carbonate and sodium hydroxide.

“Necrosis” as referred to herein is an area of dead cells. These arecommonly found in tumours due to cellular injury and premature celldeath caused by factors external to the cell or tissue, such as traumafrom inadequate supply of nutrients and oxygen.

The terms “administered”, “administration” and the like when used inreference to the administration of a compound to a target cell areintended to encompass all methods of introduction and are not intendedto be limited to direct administration to the site of a tumour cell. Theterms are intended to encompass indirect methods of introducing thecompound to the target cell for example using GDEPT, VDEPT, CDEPT orADEPT.

The phrase “therapeutically effective amount” is intended to mean anamount of a compound that has the potential to elicit a therapeuticeffect. In the case of a prodrug, it will be understood by a skilledperson that this will only actually elicit a therapeutic effect afteractivation/metabolism of that prodrug.

“Therapeutically proximate” as referred to herein in relation to abystander effect means a cell that is sufficiently close to a targetcell capable of metabolising/activating a prodrug that the cell receivestherapeutically effective concentrations of active/cytotoxic prodrugmetabolites. Without wishing to be bound by theory this is typicallywithin 1 to 10 cell diameters of the target cell.

The following is a description of the present invention, includingpreferred embodiments thereof, given in general terms. The invention isfurther elucidated from the disclosure given under the heading“Examples” herein below, which provides experimental data supporting theinvention, specific examples of various aspects of the invention, andmeans of performing the invention.

The present invention broadly relates to a new class of compounds thathave particular use as agents or drugs for cancer therapy and relatedmethods. In particular, the invention provides a specific class ofnitrobenzamide mustards, nitrobenzamide mustard alcohols and theircorresponding phosphate esters, for use as targeted cytotoxic agents orbioreductive prodrugs.

The prior art suggests that elimination of AKR1C3 activation can beachieved in a related class of dinitrobenzamide mustard prodrugs(Patterson et al, WO/2010/044685A1). The homologous N-alkylcarboxamideseries 5 to 9 (Scheme 3) was studied in isogenic HCT116 cell linescomparing cytotoxicity of the compounds in either wild-type or AKR1C3over-expressing cells in a conventional two dimension, low cell densityIC50 assay. HCT116 is a cell line derived from human colon cancer. Dataindicated that the incremental N-alkyl extension (H to methyl to ethylto isopropyl to propyl) diminished AKR1C3 dependent cell sensitivityunder aerobic conditions. It was therefore concluded that prodrugs 8 and9 were not substrates for AKR1C3.

However, the inventors have recently determined that low cell densityIC50 assays can produce ‘false negatives’. As the prodrug series 5 to 9becomes increasingly more lipophilic the inventors have found thatAKR1C3-formed metabolite is lost from the cell of production into theessentially infinite dilution of the cell culture media of the IC50assay. This loss protects the cell from cytotoxic insult such that themost lipophilic compounds 8 and 9 appear negative for AKR1C3 dependentcell sensitivity (FIG. 15). Hence, the inventors have determined throughrecombinant AKR1C3 metabolism studies that the entire series 5 to 9 isreadily metabolised by AKR1C3 (FIG. 16).

The inventors have shown that growing AKR1C3 positive cells in a threedimensional layer and then exposing this multicellular layer (MCL) toprodrugs 5 to 9 results in extensive clonogenic cell killing compared tothe AKR1C3 negative control MCL experiments (FIG. 17A). Here metaboliteproduced in one cell is lost to the neighbouring cell, exacting itscytotoxicity there. The inventors have therefore found that this highcell density MCL screen provides a method to detect ‘false negatives’from a two dimensional in vitro IC50 screen and demonstrated that it canbe used in the design of bona fide AKR1C3-negative prodrugs, such asthose of the present invention.

Therefore the disclosure of WO/2010/044685A1 is not indicative that anyform of nitrobenzamide mustard, nitrobenzamide mustard alcohol or theircorresponding phosphate ester would be resistant to metabolism by theenzyme AKR1C3. It was therefore surprising that the class of prodrugsthat are the subject of the present invention exhibited such resistanceto metabolism by AKR1C3.

The inventors have identified compounds that show reduced or zerometabolism by the human enzyme AKR1C3 when compared to known compounds(FIGS. 18 and 19). The novel compounds have the advantage that they areselectively metabolised by hypoxic tumour regions or exogenousnitroreductase expressing cells rather than being metabolised by humanAKR1C3 naturally present in other tissues such as bone marrow. Thisselectivity may be desirable to reduce side effects of the compound whenadministered to a patient. The selectivity may also reduce thetherapeutically effective dose required which has advantages includingreduced cost and potential side effects.

Previously known compounds have had to be administered in either neatDMSO or DMSO/polyethylene glycol/water (Atwell et al., J. Med. Chem.,2007, 50, 1197-1212) which results in large variations in maximumtolerated dose. An advantage of the compounds of the present inventionis their solubility in water. This has advantages for dissolution of thecompound for effective preparation of a composition of the invention andenables pharmacokinetic calculations regarding dosage and otherparameters to be measured more accurately. The increased solubility alsoprovides for more effective administration and assists with efficienttransport of the prodrug to the site of activation within the body.

In a particular embodiment, the solubility of the compound of the firstor second aspect is greater than about 95 mM when determined inPhosphate Buffered Saline (PBS) containing 2 equivalents of sodiumbicarbonate or greater than 10 mM when determined in Lactate Buffer atpH=4. The solubility of compounds 10, 11, 23 and 300 (FIG. 14) which arerepresentative of the novel class as a whole, compares to a solubilityof compound 4 of 0.068 (when determined in α-Minimal Essential Media(α-MEM) containing 5% Fetal Calf Serum (FCS)). A solubility of greaterthan 10 mM is sufficiently soluble for a drug to be useful in thiscontext. Therefore the compounds of the present invention exhibitsurprisingly appropriate solubility characteristics for use as prodrugs.

A further embodiment of the invention that is enabled by thesurprisingly high solubility of compounds of the invention is a solublecomposition comprising a compound of the invention. Such compositionsare of use in cell ablation or for the treatment of cancer and otherhyperproliferative conditions.

Compounds of the present invention comprise the nitrobenzamide mustards,nitrobenzamide mustard alcohols and their corresponding phosphateesters. The nitrobenzamide mustards are relatively inactive in theirnitro form, however on reduction are converted into a range of active(cytotoxic) compounds which can be utilised for cell ablation, forexample ablation of tumour cells.

In a particular embodiment the invention provides a method of cellablation comprising the use of a compound of the invention. In aparticular embodiment, the compound is a prodrug capable of activationby contact with a) at least one nitroreductase enzyme, and/or b) a lowoxygen (hypoxic) environment.

The inventors have also demonstrated effective methods for selecting acompound which is substantially resistant to AKR1C3 enzyme metabolism(as defined above) and use of that compound in a method of cellablation. Specifically, FIG. 16 shows compounds of the prior art thatare metabolized by AKR1C3, FIG. 17A shows how these AKR1C3 metabolisedcompounds give increased clonogenic cell kill in MCLs that over-expressAKR1C3 compared to the wild type cells and FIG. 19A shows compounds ofthe present invention that must be resistant to AKR1C3 metabolismbecause of their inability to provide increased clonogenic cell kill inmulticellular layers of cells engineered to over-express AKR1C3 relativeto multicellular layers of wild type isogenic cells.

In a further embodiment, the compound of the invention or a mixturethereof is administered to a subject in an effective amount to ablate acell wherein said cell expresses at least one nitroreductase enzyme.

The compounds of the invention are able to penetrate tumour tissue andbe selectively reduced to an active (cytotoxic) form by contact with anitroreductase enzyme (FIGS. 20-22) or by contact with a hypoxicenvironment such as that found in a tumour. This active form is able toablate the target cells and therefore has particular utility in thetreatment of cancer and other hyperproliferative disorders. In aparticular embodiment, the nitroreductase enzyme is encoded for by thenfsB and/or the nfsA gene of either E. coli or by orthologous genes inother bacterial species. In an alternative embodiment, thenitroreductase is encoded by a mutant nitroreductase. The inventionprovides a compound that may be administered to a subject in combinationwith a therapy that results in expression of an exogenous nitroreductaseenzyme within, or therapeutically proximate to, a tumour.

In the presence of pathological hypoxia found in human solid tumours,net reduction to hydroxylamine and amine cytotoxic metabolites is ableto occur providing tumour-selective cell ablation. In addition tometabolism by nitroreductase enzymes, the compounds of the presentinvention are also metabolised in hypoxic regions that may be found intumour regions (FIGS. 23-25). Thus in a further particular embodimentthe invention provides a method of cell ablation including the step ofadministering a compound of the invention or a mixture thereof in aneffective amount to ablate a cell wherein said cell is found in, ortherapeutically proximate to, a hypoxic region.

The invention therefore provides a method of treatment of cancer or ahyperproliferative condition wherein a compound of the invention or amixture thereof is administered in a therapeutically effective amount toa tumour cell, or therapeutically proximate to a tumour cell, in asubject.

Such compounds of the invention also have utility for the preparation ofa composition for the ablation of a cell, or for the treatment of canceror a hyperproliferative condition. In a particular embodiment, thecompound is a prodrug capable of activation by contact with a) at leastone nitroreductase enzyme, and/or b) a low oxygen (hypoxic) environment.

The inventors have also surprisingly found that a compound of thepresent invention, when administered to a cell in conjunction withradiation treatment is especially effective in ablating the cell (FIG.26). Accordingly, it is envisaged that the invention provides a methodof cancer treatment incorporating the administration of a prodrug of theinvention and irradiation of the tumour cells. The irradiation step maybe carried out before, concurrently with or after the administration ofthe prodrug compound.

Once metabolised, the active form of the prodrug, a cytotoxicmetabolite, is then capable of ablation of nitroreductase naive cells byway of a bystander effect. This ability to ablate cells by way of abystander effect is determined in a three dimensional cell culturemodel. This ability has particular use for the ablation of cellssurrounding nitroreductase-expressing cells in a tumour.

The inventors have previously cloned and assembled a phylogeneticallydiverse library of 55 nitroreductase candidates from 20 bacterialspecies, representing 12 different enzyme families. These bacterialnitroreductase enzymes have been screened for their ability toco-metabolise nitroimidazole imaging probes (bio-imaging) andbioreductive prodrugs (bio-therapy, bio-control) and the NfsA and NfsBfamilies have been identified as being of particular interest. Thenitroreductase enzyme may be a nitroreductase described inWO/2012/008860, which includes mutant nitroreductases and functionallyequivalent variants of the nitroreductases described therein.

The inventors have also developed novel screening methodologies toquantify the activity of a candidate nitroreductase with a targetprodrug. Further details of the screening methods used and results ofthe NTR screening of the prodrugs in bacteria and the NfsA kinetics andNfsB rate of metabolism assays are provided in Example 3 and FIGS. 17Bto 17L. Results show that the compounds tested are effectivelymetabolised by bacterial nitroreductases from a number of differentspecies and enzyme families. Therefore the compounds of the inventionhave broad-spectrum affinity to, and are substrates for, multiplebacterial nitroreductases with potential for therapeutic utility whileretaining resistance to metabolism by the endogenous human reductaseAKR1C3. The compounds selected for screening are representative of theother compound groups encompassed by the invention and similar resultswould be expected.

The compounds of the present invention are broadly defined by Formula(I) or Formula (II), where Formula (I) is:

wherein W represents Cl, Br, I, OSO₂R,

X represents Cl, Br, I, OSO₂R,

Y represents H, CN, SO₂R,

each R independently represents a lower C₁₋₆ alkyl group,

Z is selected from any of the radicals of Formula (Ia)

-   -   Where        -   R₁ represents H, or a lower C₁₋₆ alkyl group,        -   R2 and R3 may independently represent H, or a lower C1-6            alkyl group; or,        -   R2 and R3 together may be linked to form a substituted or            unsubstituted heterocyclic ring comprising 5 or 6 members,        -   n represents 2 to 6        -   * represents a point of attachment to Formula I

or a pharmaceutically acceptable salt thereof;

and where Formula (II) is:

wherein W represents Cl, Br, I, OSO₂R,

X represents Cl, Br, I, OSO₂R,

each R independently represents a lower C₁₋₆ alkyl group,

Z is selected from any of the radicals of Formula (IIa)

-   -   where        -   R₁ represents H, or a lower C₁₋₆ alkyl group,        -   n represents 2 to 6        -   * represents a point of attachment to Formula II

or a pharmaceutically acceptable salt thereof.

The compounds of Formula (I) and (II) can be used in for treating orpreventing cancer or hyperproliferative conditions. Methods of treatmentas previously described comprise the step of administering a compound ofFormula I or II, or pharmaceutically acceptable salts thereof, or amixture thereof to a subject in need thereof. Further, there is providedthe use of a compound of Formula I or II or a mixture thereof in themanufacture of a composition for the treatment of cancer orhyperproliferative conditions.

In another embodiment there is provided a method of cell ablationincluding the step of administering a compound of Formula I or II, orpharmaceutically acceptable salts thereof, or a mixture thereof in aneffective amount to ablate cells wherein said cells express at least onenitroreductase enzyme or are in a hypoxic environment.

In a further embodiment of the invention there is provided a method ofcancer treatment wherein a compound of the invention or a mixturethereof is administered in a therapeutically effective amount to atumour cell in a subject.

Preferably the cells that express the at least one nitroreductase enzymeare tumour cells in tissue in a subject.

Preferably the cells are mammalian cells.

In another embodiment there is provided a method of cancer treatmentwherein a compound of Formula I or II, or pharmaceutically acceptablesalts thereof, is administered in a therapeutically effective amount totumour cells in a subject.

Preferably the therapeutically effective amount administered is betweenabout 20% to 100% of the maximum tolerated dose of said subject.

Preferably the compound of Formula I or II, or pharmaceuticallyacceptable salts thereof, is administered for use in cell ablation inconjunction with at least one nitroreductase enzyme.

Preferably the compound of Formula I or II, or pharmaceuticallyacceptable salts thereof, or mixture thereof is administered inconjunction with GDEPT (gene-directed enzyme prodrug therapy), VDEPT(virus-directed enzyme prodrug therapy), CDEPT (clostridia-directedenzyme prodrug therapy) or ADEPT (antibody-directed enzyme prodrugtherapy).

Preferably the at least one nitroreductase enzyme is encoded for by thenfsA gene or the nfsB gene of E. coli or by orthologous genes in otherbacterial species.

Preferably the method of cancer treatment further included the step ofadministering one or more chemotherapeutic agent and/or therapies to thesubject before, during or after the administration of the compounds ofFormula I or II or mixture thereof.

While these compounds will typically be used in cancer prevention orcancer therapy of human subjects, they can be used to target cancercells in other warm blooded animal subjects, such as other primates,farm animals such as cattle, and sports animals and pets such as horses,dogs and cats.

In another embodiment there is provided a pharmaceutical compositionincluding a therapeutically effective amount of a compound of Formula(I) or Formula (II) or pharmaceutically acceptable salts thereof, or amixture thereof, and a pharmaceutically acceptable excipient, adjuvant,carrier, buffer or stabiliser.

In one embodiment the composition will be in the form of a tablet,capsule, powder, or liquid. The composition may be formulated foradministration parenterally, preferably by intravenous infusion.

In a particular embodiment, the composition is soluble in aqueoussolution. Preferably, the solubility of the compound of the first orsecond aspect as found in the composition is greater than 95 mM whendetermined in Phosphate Buffered Saline (PBS) containing 2 equivalentsof sodium bicarbonate or greater than 10 mM when determined in LactateBuffer at pH=4.

The concentration of the prodrug will depend on the nature of theprodrug used and the amount required to achieve a therapeutic effectonce activated by the nitroreductase/hypoxic environment. It will beunderstood, however, that the amount of the compound actuallyadministered will be determined by a physician, in the light of therelevant circumstances, including the condition to be treated, thechosen route of administration, the actual compound administered, theage, weight, and response of the individual patient, the severity of thepatient's symptoms, and the like, and the treatment required. In certainembodiments, the composition comprises at least one compound of theinvention in the form of pharmaceutically acceptable salts thereof, ahydrate thereof, or a solvate of any of the foregoing. Salts of theamines of the invention may include chloride, bromide, methansulfonate,tosylate, malate salts. Salts of the acids of the invention may includesodium, calcium, potassium acids wherein the acids comprise phosphateacids and carboxylic acids.

The composition of use can include a pharmaceutically acceptablediluent, carrier, buffer, stabiliser, excipient and/or adjuvant of anyof the foregoing. The choice of diluent, carrier, buffer, stabiliserexcipient and/or adjuvant can depend upon, among other factors, thedesired mode of administration. Some examples of suitable excipientsinclude lactose, dextrose, sucrose, sorbitol, mannitol, starches, gumacacia, calcium phosphate, alginates, tragacanth, gelatin, calciumsilicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose,water, syrup, and methyl cellulose. The compositions can additionallyinclude lubricating agents such as talc, magnesium stearate, and mineraloil, wetting agents, emulsifying and suspending agents, preservingagents such as methyl- and propylhydroxy-benzoates, sweetening agents,pH adjusting and buffering agents, toxicity adjusting agents, flavoringagents, and the like. The compositions can be formulated so as toprovide quick, sustained or delayed release of the active ingredientafter administration to the patient by employing procedures known in theart. A composition can be formulated in unit dosage form, each dosagecomprising a physically discrete unit suitable as a unitary dosage forhumans and other mammals, each unit containing a predetermined quantityof active material calculated to produce the desired therapeutic effect,in association with a suitable pharmaceutical excipient, diluent,carrier and/or adjuvant.

Bystander effects can be quantified according to methods described inWilson et al, 2002, Cancer Res. 62:1425-1432, by employing a 3Dmulticellular layer (MCL) composed of a minority (1%) of NTR-expressing‘activator’ cells, mixed with a majority (99%) of parental (wild-type)‘target’ cells. The prodrug concentrations for 10% survival (C₁₀) oftarget cells (wild-type cells) grown without activators (T), and targetsin co-culture (T_(C)) and activators (NTR-expressing cells) inco-culture (Ac) can be determined. The bystander effect of a testprodrug is measured by the bystander effect efficiency which can becalculated using the algorithm ((Log C₁₀T-Log C₁₀T_(C))/(Log C₁₀T-LogC₁₀A_(C))). A BEE value of less than about 15%, less than about 10%,less than about 5%, less than about 1% or zero is considered“substantially minimal”, whilst a BEE value of greater than about 50%,about 60%, about 70% is considered “substantial”.

EXAMPLES Example 1 Materials and Methods for Compound Synthesis

A series of nitrophenyl mustard prodrugs were synthesised andcharacterised by HPLC, MS, NMR and elemental analysis.

A general synthetic scheme for the synthesis of 4-alkylsulfone prodrugsof Formula I is shown in FIG. 8. As will be understood by one skilled inthe art, reaction of 3,4-difluorobenzaldehyde (100) with sodiumalkanesulfinates provides the alkylsulfones (Ill) which can be oxidisedwith sodium chlorite in phosphate buffer containing hydrogen peroxide togive the acids (IV). Nitration of these provides the nitroacids (V),which can be reacted directly with diethanolamine to give diols (VI), orfirst protected to give the tert-butyl esters (VIII), which aresubsequently reacted with diethanolamine to give diols (IX). Thionylchloride mediated chlorination of diols (VI) and subsequent reaction ofthe resulting acid chloride intermediates with aliphatic amines provides1-carboxamide dichloro mustards (VII) which can undergo lithium halidemediated halogen exchange in methyl ethyl ketone at reflux to affordcompounds of formula I. Alternately diols (IX) can be converted to theirbis-alkanesulfonate esters (X) by reaction with the appropriatealkylsulfonyl chlorides. Tert-butyl ester deprotection of thebis-alkanesulfonate esters (X) with trifluoroacetic acid affords theacids (XI). Reaction of these with oxalyl chloride in the presence ofmagnesium oxide provides the acid chloride intermediates which can befurther reacted with aliphatic amines to give the bis-alkanesulfonate1-carboxamide derivatives (XII). These can be reacted with excesslithium halide at room temperature in acetone to afford symmetricalmustards of formula I, while reaction with 1 equivalent of lithiumhalide at room temperature in acetone provides unsymmetrical haloalkanesulfonate mustards of formula I.

A preferred general synthetic scheme for the synthesis of 4-alkylsulfoneprodrugs bearing a 1-position tertiary carboxamide of Formula I is shownin FIG. 9A. As will be understood by one skilled in the art, nitroacids(V) can be converted to the acid chlorides (XIX) by reaction with oxalylchloride. Reaction of these with secondary aliphatic amines thenprovides the tertiary amides (XX) which can be reacted withdiethanolamine to give the diols (XXI). Diols (XXI) can then beconverted to their bis-alkanesulfonate esters (XII) by reaction with theappropriate alkylsulfonyl chlorides. These can be reacted with excesslithium halide at room temperature in acetone to afford symmetricalmustards of formula I, while reaction with 1 equivalent of lithiumhalide at room temperature in acetone provides unsymmetrical haloalkanesulfonate mustards of formula I.

A general synthetic scheme for the synthesis of 4-alkylsulfone prodrugsbearing acid sidechains of Formula I is shown in FIG. 9B. As will beunderstood by one skilled in the art, thionyl chloride mediatedchlorination of diols (VI) and subsequent reaction of the resulting acidchloride intermediates with aliphatic amines bearing a tert-butyl esterprotected acid sidechain, provides the 1-carboxamide dichloro mustards(XXII). Lithium halide mediated halogen exchange in methyl ethyl ketoneat reflux, followed by trifluoroacetic acid mediated ester deprotection,then provides acid sidechain bearing symmetrical mustard compounds offormula I. Alternately, reaction of the acids (XI) with oxalyl chloridein the presence of magnesium oxide provides the acid chlorides which canthen be reacted with aliphatic amines bearing a tert-butyl esterprotected acid sidechain to provide the 1-carboxamidebis-alkanesulfonate mustards (XXIII). Reaction of these with excesslithium halide at room temperature in acetone, followed bytrifluoroacetic acid mediated ester deprotection then provides acidsidechain bearing compounds with symmetrical mustards of formula I.Reaction of the 1-carboxamide bis-alkanesulfonate mustards (XXIII) with1 equivalent of lithium halide at room temperature in acetone, followedby trifluoroacetic acid mediated ester deprotection, then provides acidsidechain bearing unsymmetrical halo alkanesulfonate mustards of formulaI.

A general synthetic scheme for synthesis of 4-cyano prodrugs of FormulaI is shown in FIG. 9C. As will be understood by one skilled in the art,reaction of 3,4-difluorobenzaldehyde (100) with sodium cyanide provides3-fluoro-4-cyanobenzaldehyde (371) which can be oxidised with sodiumchlorite in phosphate buffer containing hydrogen peroxide to give the3-fluoro-4-cyanobenzoic acid (372). Nitration and subsequenttrifluoroacetic anhydride mediated dehydration of the resultantcarboxamide and aqueous basic work-up provides acid (373) which isprotected as the tert-butyl ester (374). Reaction with diethanolaminegives diol (375), which can be converted to the bis-alkanesulfonateesters (XXIV) by reaction with the appropriate alkylsulfonyl chlorides.Tert-butyl ester deprotection of the bis-alkanesulfonate esters (XXIV)with trifluoroacetic acid affords the acids (XXV). Reaction of thesewith oxalyl chloride in the presence of magnesium oxide provides theacid chloride intermediates which can be further reacted with aliphaticamines to give the bis-alkanesulfonate 1-carboxamide derivatives (XXVI).These can be reacted with excess lithium halide at room temperature inacetone to afford symmetrical mustards of formula I, while reaction with1 equivalent of lithium halide at room temperature in acetone providesunsymmetrical halo alkanesulfonate mustards of formula I.

A preferred general synthetic scheme for the synthesis of 4-cyanoprodrugs bearing a 1-position tertiary carboxamide of Formula I is shownin FIG. 9D. As will be understood by one skilled in the art, acid (373)can be converted to the acid chloride (376) by reaction with oxalylchloride. Reaction of this with secondary aliphatic amines then providesthe teritary amides (XXVII) which can be reacted with diethanolamine togive the diols (XXVIII). Diols (XXVIII) can then be converted to theirbis-alkanesulfonate esters (XXVI) by reaction with the appropriatealkylsulfonyl chlorides. These can be reacted with excess lithium halideat room temperature in acetone to afford symmetrical mustards of formulaI, while reaction with 1 equivalent of lithium halide at roomtemperature in acetone provides unsymmetrical halo alkanesulfonatemustards of formula I.

A general synthetic scheme for the synthesis of 4-cyano prodrugs bearingacid sidechains of Formula I is shown in FIG. 9E. As will be understoodby one skilled in the art, reaction of the acids (XXV) with oxalylchloride in the presence of magnesium oxide, provides the acid chlorideswhich can then be reacted with aliphatic amines bearing a tert-butylester protected acid sidechain to provide the 1-carboxamidebis-alkanesulfonate mustards (XXIX). Reaction of these with excesslithium halide at room temperature in acetone, followed bytrifluoroacetic acid mediated ester deprotection then provides acidsidechain bearing compounds with symmetrical mustards of formula I.Reaction of the 1-carboxamide bis-alkanesulfonate mustards (XXIX) with 1equivalent of lithium halide at room temperature in acetone, followed bytrifluoroacetic acid mediated ester deprotection, then provides acidsidechain bearing unsymmetrical halo alkanesulfonate mustards of formulaI.

A general scheme for synthesis of prodrugs of Formula II is shown inFIG. 12. As will be understood by one skilled in the art, reaction of2-fluoro-5-nitrobenzoic acid (125) with thionyl chloride and subsequentreaction of the resulting acid chloride with aliphatic amines bearingTHP protected alcohols provides carboxamides (XIII). Reaction withdiethanolamine then gives diols (XIV), which can be converted to theirbis-alkanesulfonate esters (XV) by reaction with the appropriatealkylsulfonyl chlorides. THP acetal deprotection of thebis-alkanesulfonate esters (XV) with the appropriate alkylsulfonic acidaffords the alcohols (XVI). These can be directly reacted with excesslithium halide at room temperature in acetone to afford symmetricalmustard alcohols of formula II, while reaction with 1 equivalent oflithium halide at room temperature in acetone provides unsymmetricalhalo alkanesulfonate mustard alcohols of formula II. These alcohols offormula II can then be converted to their respective phosphates by firstreacting them with with di-tert-butyl-N,N-diisopropylphosphoramidite inthe presence of 1H-tetrazole, followed by oxidation withmeta-chloroperoxybenzoic acid to give the intermediatetert-butylphosphate esters XVIII and XVII, respectively. Deprotection ofthese with trifluoroacetic acid in dichloromethane then gives thephosphates of formula II.

A scheme for synthesis of alcohol compound 14 (FIG. 2) is shown in FIG.9F. Reaction of 3,4-difluorobenzaldehyde (100) with sodiummethylsulfinate gave methylsulfone 101, which was oxidised with sodiumchlorite in phosphate buffer containing hydrogen peroxide to give acid102. Nitration gave acid 103, which was reacted directly withdiethanolamine to give diol 104, or first protected to give thetert-butyl ester 106, which was subsequently reacted with diethanolamineto give diol 107. Thionyl chloride mediated chlorination of diol 104 andsubsequent reaction of the resulting acid chloride intermediate with2-(methylamino)ethanol gave the dichloro mustard 111 which was subjectedto lithium bromide mediated halogen exchange in methyl ethyl ketone atreflux to afford compound 14. Alternately diol 107 was converted to thebis-methanesulfonate ester 108 by reaction with methanesulfonylchloride. Tert-butyl ester deprotection of bis-methanesulfonate ester108 with trifluoroacetic acid gave acid 109. Reaction of this withoxalyl chloride in the presence of magnesium oxide provided the acidchloride intermediate which was further reacted with2-(methylamino)ethanol to give the bis-methanesulfonate 1-carboxamide112. Reaction of this with excess lithium bromide at room temperature inacetone gave compound 14.

A scheme for synthesis of alcohol compound 18 (FIG. 2) is shown in FIG.9G. Reaction of 3,4-difluorobenzaldehyde (100) with sodiumethylsulfinate gave ethylsulfone 113, which was oxidised with sodiumchlorite in phosphate buffer containing hydrogen peroxide to give acid114. Nitration gave acid 115, which was protected to give the tert-butylester 116. Subsequently reaction with diethanolamine gave diol 117 whichwas converted to the bis-methanesulfonate ester 118 by reaction withmethanesulfonyl chloride. Tert-butyl ester deprotection ofbis-methanesulfonate ester 118 with trifluoroacetic acid gave acid 119.Reaction of this with oxalyl chloride in the presence of magnesium oxideprovided the acid chloride intermediate which was further reacted with2-(methylamino)ethanol to give the bis-methanesulfonate 1-carboxamide120. Reaction of this with excess lithium bromide at room temperature inacetone gave compound 18.

A scheme for synthesis of alcohol compound 301 (FIG. 2) is shown in FIG.9H. Thionyl chloride mediated chlorination of diol 104 and subsequentreaction of the resulting acid chloride intermediate with 1-aminoethanolgave the dichloro mustard 303 which was subjected to lithium bromidemediated halogen exchange in methyl ethyl ketone at reflux to affordcompound 301. Alternately bis-methanesulfonate ester 109 was reactedwith oxalyl chloride in the presence of magnesium oxide to give the acidchloride intermediate which was further reacted with 1-aminoethanol togive the bis-methanesulfonate 1-carboxamide 304. Reaction of this withexcess lithium bromide at room temperature in acetone gave compound 301.

A scheme for synthesis of phosphates 10, and 11 and 300 (FIG. 1) isshown in FIG. 10. Reaction of alcohols 14, 18 and 301 withdi-tert-butyl-N,N-diisopropylphosphoramidite in the presence of1H-tetrazole, followed by oxidation with meta-chloroperoxybenzoic acidgave the tert-butylphosphate esters 122, 123 and 302 respectively.Deprotection of these with trifluoroacetic acid in dichloromethane gavethe phosphates 10, 11 and 300 respectively.

A scheme for synthesis of prodrugs 22 to 27 (FIG. 3) is shown in FIG.11. Reaction of bis-methanesulfonate ester 109 with oxalyl chloride inthe presence of magnesium oxide gave the acid chloride intermediatewhich was further reacted with 1-methylpiperazine to give thebis-methanesulfonate 1-carboxamide 124. Reaction of this with excesslithium bromide at room temperature in acetone gave compound 22. Thionylchloride mediated chlorination of diol 104 and subsequent reaction ofthe resulting acid chloride intermediate with 1-ethylpiperazine and1-iso-propylpiperazine gave the dichloro mustards 131 and 132,respectively. Lithium bromide mediated halogen exchange in methyl ethylketone at reflux then gave compounds 23 and 24, respectively. Reactionof bis-methanesulfonate ester 119 with oxalyl chloride in the presenceof magnesium oxide gave the acid chloride intermediate which was furtherreacted with 1-methylpiperazine, 1-ethylpiperazine and1-iso-propylpiperazine to give the bis-methanesulfonate 1-carboxamides133, 134 and 135, respectively. Reaction of these with excess lithiumbromide at room temperature in acetone gave compounds 25, 26 and 27,respectively.

A scheme for synthesis of alcohol 64 and phosphate 60 is shown in FIG.13.

Reaction of 2-fluoro-5-nitrobenzoic acid 125 with thionyl chloride andsubsequent reaction of the resulting acid chloride with ethanolaminegave amide 136. Subsequent THP-protection of this with3,4-dihydro-2H-pyran in the presence of catalytic para-toluenesulfonicacid gave amide 126. This could be directly prepared by reaction of thepreviously described acid chloride with THP-protected ethanolamine.Reaction of amide 126 with diethanolamine then gave diol 127, which wasconverted to bis-methanesulfonate ester 128 by reaction withmethanesulfonyl chloride. THP acetal deprotection with themethanesulfonic acid gave alcohol 129, which reacted with 1 equivalentof lithium bromide at room temperature in acetone to give compound 64.Reaction of this with di-tert-butyl-N,N-diisopropylphosphoramidite inthe presence of 1H-tetrazole, followed by oxidation withmeta-chloroperoxybenzoic acid gave tert-butylphosphate ester 130 whichwas deprotected employing trifluoroacetic acid in dichloromethane togive phosphate 60.

Solubility and stability of compounds in media (+5% Fetal calf serum),phosphate buffered saline (+sodium bicarbonate) or lactate buffer (atpH=4) were determined by HPLC.

Phosphate ester compounds typically exhibit superior aqueous solubilitythan the alcohol compounds they are derived from and are used here aspre-prodrugs. They are known to be rapidly cleaved in plasma by serumphosphatases to release the alcohol derivatives.

5-(bis(2-bromoethyl)amino)-N-(2-hydroxyethyl)-N-methyl-4-(methylsulfonyl)-2-nitrobenzamide(14)

Method 1

3-fluoro-4-(methylsulfonyl)benzaldehyde (101)

3,4-Difluorobenzaldehyde 100 (10.00 g, 70.37 mmol) was treated withsodium methanesulfinate (10.06 g, 98.53 mmol) in DMSO (200 mL) at theroom temperature. The reaction mixture was heated at 75° C. under N₂ for3 h then cooled to the room temperature, and poured into a beaker ofice-water. The white solid was collected by filtration, washed withwater, and dried in a vacuum oven at 45° C. The crude product waspurified by flash column chromatography on silica gel eluting withCH₂Cl₂/hexane (4:1) then neat CH₂Cl₂ to give3-fluoro-4-(methylsulfonyl)benzaldehyde 101 (11.83 g, 83%) as a whitepowder. M.p. and ¹HNMR consistent with the desired product. Note: At 75°C. predominantly the desired isomer is formed. At temperature above 75°C. for example 90° C., the ratio of the mono- to bis-methylsulfonyl is2.4:1.

3-fluoro-4-(methylsulfonyl)benzoic acid (102)

To a solution of 3-fluoro-4-(methylsulfonyl)benzaldehyde 101 (11.50 g,56.87 mmol) in CH₃CN (105 mL) at the room temperature, a buffer solutionof NaH₂PO₄.4H₂O (1.86 g, 9.69 mmol) and conc. HCl (1.2 mL) in water(39.1 mL) and then H₂O₂ (35%, 9.7 mL, 285.21 mmol) were added. Thereaction mixture was cooled to 0° C. and a solution of NaClO₂ (7.21 g,79.72 mmol) in water (133 mL) was added dropwise. After stirring at roomtemperature for 5 h, the solvents were removed to half a volume and thewhite solid was collected by filtration. The filtrate was treated withconc. HCl and some more products were precipitated and collected byfiltration. The combined solid was dried in a vacuum oven at 45° C. togive 3-fluoro-4-(methylsulfonyl)benzoic acid 102 (12.31 g, 99%) as awhite powder. M.p. and ¹HNMR consistent with the desired product.

5-fluoro-4-(methylsulfonyl)-2-nitrobenzoic acid (103)

3-Fluoro-4-(methylsulfonyl)benzoic acid 102 (13.00 g, 59.58 mmol) wasdissolved in conc. H₂SO₄ (93 mL) and fuming HNO₃ (18 mL) was addeddropwise at the room temperature. The reaction mixture was heated at 45°C. for 4 h, cooled to the room temperature and poured into a beaker ofice-water. The solid was collected by filtration, washed several timeswith water, and dried in a vacuum oven at 45° C. to provide5-fluoro-4-(methylsulfonyl)-2-nitrobenzoic acid 103 (12.14 g, 77%) as apale yellow solid. M.p. and ¹HNMR consistent with the desired product.

5-(Bis(2-hydroxyethyl)amino)-4-(methylsulfonyl)-2-nitrobenzoic acid(104)

Method 1

5-Fluoro-4-(methylsulfonyl)-2-nitrobenzoic acid 103 (3.00 g, 11.40 mmol)was dissolved in DMSO (30 mL) and treated with diethanol amine (3.27 mL,34.12 mmol) at room temperature. The reaction mixture was heated at 45°C. for 2 h, cooled to the room temperature and poured into a beaker ofice-water. The crude yellow gum was extracted with EtOAc/i-PrOH (4:1)(3×) and the combined organic phases were washed with water (6×), driedwith Na₂SO₄ and concentrated under reduced pressure (water bath 35° C.)to give 5-(bis(2-hydroxyethyl)amino)-4-(methylsulfonyl)-2-nitrobenzoicacid 104 (3.64 g, 92%) as a yellow gum. ¹HNMR [(CD₃)₂SO] δ 14.07 (br, s,1H), 8.49 (s, 1H), 7.69 (s, 1H), 4.61 (br, s, 2H), 3.57-3.54 (m, 4H),3.51-3.48 (m, 4H), 3.46 (s, 3H). HRMS (APCI) calcd for C₁₂H₁₇N₂O₈S[M+H]⁺ m/z 349.0705: found 349.0687.

5-(Bis(2-hydroxyethyl)amino)-4-(methylsulfonyl)-2-nitrobenzoic acid(104)

Method 2

5-Fluoro-4-(methylsulfonyl)-2-nitrobenzoic acid 103 (7.80 g, 29.64 mmol)was dissolved in DMSO (25 mL) and treated with diethanol amine (8.51 mL,88.79 mmol). The reaction mixture was stirred at room temperature for 2h then poured into a beaker of ice-cold aqueous HCl (2M, 100 mL),extracted with EtOAc/i-PrOH (4:1) (3×), washed with brine, dried withNa₂SO₄ and concentrated under reduced pressure to give5-(bis(2-hydroxyethyl)amino)-4-(methylsulfonyl)-2-nitrobenzoic acid 104(8.08 g, 78%) as a yellow powder.

5-(Bis(2-chloroethyl)amino)-N-(2-hydroxyethyl)-N-methyl-4-(methylsulfonyl)-2-nitrobenzamide(111)

A stirred solution of5-(bis(2-hydroxyethyl)amino)-4-(methylsulfonyl)-2-nitrobenzoic acid 104(1.0 g, 2.87 mmol) in SOCl₂ (25 mL) and DMF (3 drops) was heated underreflux for 4 h. The excess SOCl₂ was removed by distillation underreduced pressure and the residue was dissolved in CH₂Cl₂ (10 mL) and THF(6 mL), cooled to 0° C. and treated with 2-(methylamino)ethanol (822

L, 10.25 mmol). The reaction mixture was stirred at 0° C. for 20 minthen warmed to the room temperature, acidified with aqueous HCl (0.5 M,8 mL) and extracted with EtOAc (2×). The combined organic phases werewashed with brine, dried with Na₂SO₄ and evaporated to dryness underreduce pressure. The crude product was purified by flash columnchromatography on silica gel eluting with CH₂Cl₂/MeOH (20:1) to give5-(bis(2-chloroethyl)amino)-N-(2-hydroxyethyl)-N-methyl-4-(methylsulfonyl)-2-nitrobenzamide111 (390 mg, 31%) as a mixture of atropisomers, as a yellow gum. ¹HNMR[(CD₃)₂SO] δ 8.66 (s, 0.4H), 8.64 (s, 0.6H), 7.71 (s, 0.6H), 7.66 (s,0.4H), 4.83-4.78 (2t, J=5.4 Hz, 1H), 3.78 (br, s, 4H), 3.76-3.71 (m,4H), 3.69-3.64 (m, 1H), 3.55-3.52 (m, 1H), 3.48 (s, 1.6H), 3.47 (s,1.4H), 3.19 (br, s, 2H), 3.04 (s, 1.6H), 2.85 (s, 1.4H). HRMS(ESI) calcdfor C₁₅H₂₁Cl₂N₃NaO₆S [M+Na]⁺ m/z 464.0407: found 464.0420.

5-(Bis(2-bromoethyl)amino)-N-(2-hydroxyethyl)-N-methyl-4-(methylsulfonyl)-2-nitrobenzamide(14)

A solution of(5-(bis(2-chloroethyl)amino)-N-(2-hydroxyethyl)-N-methyl-4-(methylsulfonyl)-2-nitrobenzamide111 (320 mg, 0.72 mmol) in 3-methyl-2-butanone (13 mL) was treated withLiBr (1.26 g, 14.51 mmol) and heated to reflux overnight. The reactionmixture was cooled to the room temperature and the solvent was removedunder reduced pressure. The residue was dissolved in EtOAc and washedwith water (3×), dried with Na₂SO₄ and concentrated under reducedpressure. The crude mixture was resubmitted to LiBr (2×) and worked upas above. The final product was purified by flash column chromatographyon silica gel eluting with CH₂Cl₂/MeOH (20:1) and further recrystallizedfrom CH₂Cl₂/iPr₂O to give5-(bis(2-bromoethyl)amino)-N-(2-hydroxyethyl)-N-methyl-4-(methylsulfonyl)-2-nitrobenzamide14 (258 mg, 67%) as a mixture of atropisomers, as a pale yellow solid:m.p. 138-140° C.; ¹HNMR [(CD₃)₂SO] δ 8.65 (s, 0.4H), 8.64 (s, 0.6H),7.72 (s, 0.6H), 7.67 (s, 0.4H), 4.82-4.77 (2t, J=5.5 Hz, 1H), 3.84-3.77(m, 4H), 3.69-3.59 (m, 5H), 3.58-3.52 (m, 1H), 3.49 (s, 1.6H), 3.48 (s,1.4H), 3.19 (br, s, 2H), 3.04 (s, 1.6H), 2.86 (s, 1.4H). Anal. calcd forC₁₅H₂₁Br₂N₃O₆S.0.2iPr₂O: C, 35.30; H, 4.28; N, 7.62%; found: C, 35.06;H, 4.09; N, 7.64%.

5-(bis(2-bromoethyl)amino)-N-(2-hydroxyethyl)-N-methyl-4-(methylsulfonyl)-2-nitrobenzamide(14)

Method 2

tert-Butyl 5-fluoro-4-(methylsulfonyl)-2-nitrobenzoate (106)

5-Fluoro-4-(methylsulfonyl)-2-nitrobenzoic acid 103 (8.24 g, 31.31 mmol)was dissolved in CH₃CN (48 mL) at 50° C., cooled to the room temperatureand treated with tert-butyl acetate (48 mL) and perchloric acid (70%,2.64 mL, 43.83 mmol). The reaction mixture was stirred at roomtemperature for 48 h and the solvents were removed under reducedpressure (water bath 50° C.). The residue was recrystallized fromMeOH/water in the cold room. The product was collected by filtration togive tert-butyl 5-fluoro-4-(methylsulfonyl)-2-nitrobenzoate 106 (5.70 g,57%) as pale yellow crystals: m.p. 99-101° C.; ¹HNMR (CDCl₃) δ 8.58 (d,J=5.7 Hz, 1H), 7.57 (d, J=8.5 Hz, 1H), 3.30 (s, 3H), 1.60 (s, 9H). Anal.calcd for C₁₂H₁₄FNO₆S: C, 45.14; H, 4.42; N, 4.39%; found: C, 45.44; H,4.47; N, 4.32%. Note: The filtrate was diluted with water and treatedwith aqueous HCl (4 M) to recover some unreacted starting material (3.09g) that was collected by filtration.

tert-Butyl5-(bis(2-hydroxyethyl)amino)-4-(methylsulfonyl)-2-nitrobenzoate (107)

A solution of tert-butyl 5-fluoro-4-(methylsulfonyl)-2-nitrobenzoate 106(6.64 g, 20.79 mmol) in DMSO (15 mL) was treated with diethanol amine(2.79 mL, 29.12 mmol). The reaction mixture was stirred at roomtemperature for 2 h then poured into a beaker of ice-water, extractedwith diethyl ether (3×), dried with Na₂SO₄ and concentrated underreduced pressure. The yellow gum was purified by flash columnchromatography on silica gel eluting with CH₂Cl₂/MeOH (19:1) to givetert-butyl5-(bis(2-hydroxyethyl)amino)-4-(methylsulfonyl)-2-nitrobenzoate 107(7.35 g, 87%) as a yellow gum.

¹HNMR [(CD₃)₂SO]δ 8.50 (s, 1H), 7.63 (s, 1H), 4.64 (t, J=5.0 Hz, 2H),3.58-3.54 (m, 4H), 3.52-3.50 (m, 4H), 3.45 (s, 3H), 1.53 (s, 9H).HRMS(ESI) calcd for C₁₆H₂₅N₂O₈S [M+H]⁺ m/z 405.1347: found 405.1326.Note: At temperature higher than room temperature, for example 40 to 50°C. a significant amount of the tert-butyl5-(2-((2-hydroxyethyl)amino)ethoxy)-4-(methylsulfonyl)-2-nitrobenzoateproduct is formed (i.e. O-alkylation in competition with N-alkylation).

tert-Butyl5-(bis(2-((methylsulfonyl)oxy)ethyl)amino)-4-(methylsulfonyl)-2-nitrobenzoate(108)

To a solution of tert-butyl5-(bis(2-hydroxyethyl)amino)-4-(methylsulfonyl)-2-nitrobenzoate 107(8.52 g, 21.07 mmol) in CH₂Cl₂ (290 mL) and Et₃N (10.28 mL, 73.75 mmol)at 0° C. MsCl (4.90 mL, 63.22 mmol) was added dropwise. The reactionmixture was stirred for 20 min at 0° C. then warmed to the roomtemperature, diluted with CH₂Cl₂, washed with water (3×), dried withNa₂SO₄ and concentrated under reduced pressure. The residues waspurified by flash column chromatography on silica gel eluting withCH₂Cl₂/MeOH (19:1) to give tert-butyl5-(bis(2-((methylsulfonyl)oxy)ethyl)amino)-4-(methylsulfonyl)-2-nitrobenzoate108 (10.60 g, 90%) as a yellow gum. ¹HNMR [(CD₃)₂SO] δ 8.52 (s, 1H),7.83 (s, 1H), 4.36 (t, J=5.2 Hz, 4H), 3.77 (t, J=5.0 Hz, 4H), 3.43 (s,3H), 3.14 (s, 6H), 1.53 (s, 9H). HRMS(ESI) calcd forC₁₈H₂₈N₂NaO₁₂S₃[M+Na]⁺ m/z 583.0674: found 583.0697.

5-(Bis(2-((methylsulfonyl)oxy)ethyl)amino)-4-(methylsulfonyl)-2-nitrobenzoicacid (109)

tert-Butyl5-(bis(2-((methylsulfonyl)oxy)ethyl)amino)-4-(methylsulfonyl)-2-nitrobenzoate108 (10.60 g, 18.91) in CH₂Cl₂ (56 mL) was treated with TFA (21 mL) at5° C. The reaction was stirred at the room temperature for 2 h, and thesolvents were removed under reduced pressure. The residue was thendissolved in EtOAc and the solvent was evaporated to dryness to removethe excess TFA. The yellow residue was dissolved in CH₂Cl₂ andprecipitated with iPr₂O to give5-(bis(2-((methylsulfonyl)oxy)ethyl)amino)-4-(methylsulfonyl)-2-nitrobenzoicacid 109 (9.54 g, 100%) as a yellow gum. ¹HNMR [(CD₃)₂SO] δ 8.50 (s,1H), 7.89 (s, 1H), 4.35 (t, J=5.1 Hz, 4H), 3.75 (t, J=5.2 Hz, 4H), 3.44(s, 3H), 3.14 (s, 6H). HRMS(ESI) calcd for C₁₄H₂₀N₂NaO₁₂S₃[M+Na]⁺ m/z527.0062: found 527.0071.

((5-((2-Hydroxyethyl)(methyl)carbamoyl)-2-(methylsulfonyl)-4-nitrophenyl)azanediyl)bis(ethane-2,1-diyl)dimethanesulfonate (112)

A solution of5-(bis(2-((methylsulfonyl)oxy)ethyl)amino)-4-(methylsulfonyl)-2-nitrobenzoicacid 109 (2.66 g, 5.27 mmol) in CH₂Cl₂ (80 mL) and CH₃CN (20 mL) wastreated with MgO (3.19 g, 79.09 mmol) at the room temperature thencooled to 0° C. and treated with oxalyl chloride (2.71 mL, 31.62 mmol)and DMF (3 drops). The reaction mixture was stirred at 0° C. for 1 hthen warmed to the room temperature for 3 h. The mixture was filteredthrough a short pad of Celite and the solvents were removed underreduced pressure. The residue was dissolved in CH₂Cl₂ (80 mL) and THF(20 mL), cooled to 0° C. and treated with 2-(methylamino)ethanol (1.27mL, 15.85 mmol) and warm to the room temperature for 20 min. The mixturewas washed with water (3×), dried with Na₂SO₄ and concentrated underreduced pressure. The residues was purified by flash columnchromatography on silica gel eluting with CH₂Cl₂/MeOH (19:1) to give((5-((2-hydroxyethyl)(methyl)carbamoyl)-2-(methylsulfonyl)-4-nitrophenyl)azanediyl)bis(ethane-2,1-diyl)dimethanesulfonate 112 (2.20 g, 74%) as a mixture of atropisomers, as ayellow gum. ¹HNMR [(CD₃)₂SO] δ 8.65 (s, 0.5H), 8.64 (s, 0.5H), 7.72 (s,0.5H), 7.67 (s, 0.5), 4.84-4.79 (2t, J=5.2 Hz, 1H), 4.36-4.34 (m, 4H),3.79-3.76 (m, 4H), 3.68-3.53 (m, 2H), 3.44 (s, 3H), 3.34-3.29 (m, 2H),3.14 (s, 6H), 3.04 (s, 1.6H), 2.86 (s, 1.4H). HRMS(ESI) calcd forC₁₇H₂₇N₃NaO₁₂S₃[M+Na]⁺ m/z 584.0647: found 584.0649.

5-(Bis(2-bromoethyl)amino)-N-(2-hydroxyethyl)-N-methyl-4-(methylsulfonyl)-2-nitrobenzamide(14)

((5-((2-Hydroxyethyl)(methyl)carbamoyl)-2-(methylsulfonyl)-4-nitrophenyl)azanediyl)bis(ethane-2,1-diyl)dimethanesulfonate 112 (3.70 g, 6.59 mmol) was dissolved in acetone (200mL) and treated with LiBr (11.44 g, 131.72 mmol) at the roomtemperature. The reaction mixture was stirred overnight and the solventwas removed. The residue was dissolved in EtOAc and washed with water(2×), dried with Na₂SO₄ and concentrated under reduced pressure. Thecrude product was purified by flash column chromatography on silica geleluting with CH₂Cl₂/MeOH (20:1) to give5-(bis(2-bromoethyl)amino)-N-(2-hydroxyethyl)-N-methyl-4-(methylsulfonyl)-2-nitrobenzamide14 (3.13 g, 89%) as a mixture of atropisomers, as a yellow solid. M.p.and ¹HNMR identical to that previously observed.

5-(Bis(2-bromoethyl)amino)-4-(ethylsulfonyl)-N-(2-hydroxyethyl)-N-methyl-2-nitrobenzamide(18) 4-(Ethylsulfonyl)-3-fluorobenzaldehyde (113)

3,4-Difluorobenzaldehyde 100 (23.50 g, 165.38 mmol) was treated withsodium ethanesulfinate (23.00 g, 198.09 mmol) in DMSO (230 mL) at theroom temperature. The reaction mixture was heated at 75° C. under N₂ for4 h then cooled to the room temperature, and poured into a beaker ofice-water. The white precipitates were collected by filtration, washedwith water, and dried in a vacuum oven at 45° C. The solid was thenrecrystallized from CH₂Cl₂/iPr₂O to give4-(ethylsulfonyl)-3-fluorobenzaldehyde 113 (28.48 g, 80%) as a whitepowder: m.p. 107-110° C.; ¹HNMR (CDCl₃) δ 10.09 (d, J=1.9 Hz, 1H), 8.16(dd, J=6.5 Hz, 1.4, 1H), 7.87 (dd, J=7.9 Hz, 1.4, 1H), 7.75 (dd, J=9.4Hz, 1.4, 1H), 3.38 (2q, J=7.4 Hz, 2H), 1.33 (2t, J=7.5 Hz, 3H). Anal.calcd for C₉H₉FO₃S: C, 49.99; H, 4.20; F, 8.79%; found: C, 50.19; H,4.23; F, 8.91%.

4-(Ethylsulfonyl)-3-fluorobenzoic acid (114)

To a solution of 4-(ethylsulfonyl)-3-fluorobenzaldehyde 113 (30.70 g,141.98 mmol) in CH₃CN (280 mL) at the room temperature, a buffersolution of NaH₂PO₄.4H₂O (4.65 g, 24.21 mmol) and conc. HCl (3.2 mL) inwater (105 mL) and then H₂O₂ (35%, 24.1 mL, 708.62 mmol) were added. Thereaction mixture was cooled to 0° C. and a solution of NaClO₂ (17.98 g,198.81 mmol) in water (350 mL) was added dropwise. After stirring atroom temperature for 5 h, the solvents were removed to half a volumeunder reduced pressure and the white solid was collected by filtration.The filtrate was treated with conc. HCl and some more products wereprecipitated and collected by filtration. The combined solid was driedin a vacuum oven at 45° C. to give 4-(ethylsulfonyl)-3-fluorobenzoicacid 114 (32.65 g, 99%) as a white powder: m.p. 184-186° C.; ¹HNMR(CDCl₃) δ 8.12-8.07 (m, 2H), 7.98-7.95 (m, 1H), 3.38 (q, J=7.4 Hz, 2H),1.34 (t, J=7.4 Hz, 3H). Anal. calcd for C₉H₉FO₄S: C, 46.55; H, 3.91; F,8.18%; found: C, 46.82; H, 3.99; F, 8.33%.

4-(Ethylsulfonyl)-5-fluoro-2-nitrobenzoic acid (115)

4-(Ethylsulfonyl) 3-fluorobenzoic acid 114 (15.65 g, 67.39 mmol) wasdissolved in conc. H₂SO₄ (107 mL) and fuming HNO₃ (21 mL) was addeddropwise at the room temperature. The reaction mixture was heated at 45°C. for 4 h, cooled to the room temperature and poured into a beaker ofice-water. The solid was collected by filtration, washed with water, anddried in a vacuum oven at 45° C. to provide4-(ethylsulfonyl)-5-fluoro-2-nitrobenzoic acid 115 (16.63 g, 89%) as apale yellow solid: m.p. 140-143° C.; ¹HNMR [(CD₃)₂SO] δ 14.59 (br, s,1H), 8.41 (d, J=5.8 Hz, 1H), 8.07 (d, J=9.3 Hz, 1H), 3.53 (q, J=7.4 Hz,2H), 1.20 (t, J=7.4 Hz, 3H). HRMS(ESI) calcd for C₉H9FNO₆S [M+1]+m/z278.0130: found 278.0129.

tert-Butyl 4-(Ethylsulfonyl)-5-fluoro-2-nitrobenzoate (116)

4-(Ethylsulfonyl)-5-fluoro-2-nitrobenzoic acid 115 (24.35 g, 87.83 mmol)was dissolved tert-butyl acetate (150 mL) and treated with perchloricacid (70%, 3.70 mL, 61.48 mmol). The reaction mixture was stirred atroom temperature overnight then further treated with perchloric acid(70%, 3.70 mL, 61.48 mmol) and left stirring for 24 h. The solvent wasthen removed under reduced pressure (water bath 50° C.), and the residuewas recrystallized from MeOH/water in the cold room. The product wascollected by filtration and further recrystallized from CH₂Cl₂/iPr₂O togive tert-butyl 4-(ethylsulfonyl)-5-fluoro-2-nitrobenzoate 116 (20.51 g,70%) as pale yellow crystals: m.p. 105-107° C.; ¹HNMR (CDCl₃) δ 8.54 (d,J=5.7 Hz, 1H), 7.54 (d, J=8.5 Hz, 1H), 3.37 (2q, J=7.6 Hz, 2H), 1.59 (s,9H), 1.37 (2t, J=7.4 Hz, 3H). Anal. calcd for C₁₃H₁₆FNO₆S.0.1iPr₂O: C,47.58; H, 5.05; N, 4.08%; found: C, 47.35; H, 4.87; N, 4.17%.

tert-Butyl5-(bis(2-hydroxyethyl)amino)-4-(ethylsulfonyl)-2-nitrobenzoate (117)

A solution of tert-butyl 4-(ethylsulfonyl)-5-fluoro-2-nitrobenzoate 116(13.15 g, 39.45 mmol) in DMSO (30 mL) was treated with diethanol amine(5.06 mL, 52.81 mmol). The reaction mixture was stirred at roomtemperature for 2 h then poured into a beaker of ice-water, extractedwith diethyl ether (3×), dried with Na₂SO₄ and concentrated underreduced pressure. The yellow gum was purified by flash columnchromatography on silica gel eluting with CH₂Cl₂/MeOH (49:1) to givetert-butyl5-(bis(2-hydroxyethyl)amino)-4-(ethylsulfonyl)-2-nitrobenzoate 117(12.90 g, 78%) as a yellow gum. ¹HNMR [(CD₃)₂SO]δ 8.48 (s, 1H), 7.64 (s,1H), 4.64 (t, J=4.8 Hz, 2H), 3.71 (q, J=7.3, 2H), 3.57-3.49 (m, 8H),1.53 (s, 9H), 0.10 (t, J=7.3 Hz, 3H). HRMS(ESI) calcd for C₁₇H₂₇N₂O₈S[M+H]⁺ m/z 419.1483: found 419.1483.

tert-Butyl5-(Bis(2-((methylsulfonyl)oxy)ethyl)amino)-4-(ethylsulfonyl)-2-nitrobenzoate(118)

To a solution of tert-Butyl5-(bis(2-hydroxyethyl)amino)-4-(ethylsulfonyl)-2-nitrobenzoate 117(12.90 g, 30.83 mmol) in CH₂Cl₂ (500 mL) and Et₃N (15.04 mL, 107.90mmol) at 0° C. MsCl (7.20 mL, 92.93 mmol) was added dropwise. Thereaction mixture was stirred for 20 min at 0° C. then warmed to the roomtemperature, diluted with CH₂Cl₂, washed with water (3×), dried withNa₂SO₄ and concentrated under reduced pressure. The residues waspurified by flash column chromatography on silica gel eluting withEtOAc/hexane (4:1) to give tert-butyl5-(bis(2-((methylsulfonyl)oxy)ethyl)amino)-4-(ethylsulfonyl)-2-nitrobenzoate118 (14.80 g, 84%) as a yellow powder. ¹HNMR [(CD₃)₂SO] δ 8.50 (s, 1H),7.81 (s, 1H), 4.36 (t, J=5.0 Hz, 4H), 3.77 (t, J=5.0 Hz, 4H), 3.64 (q,J=7.4 Hz, 2H), 3.15 (s, 6H), 1.53 (s, 9H), 1.08 (t, J=7.3 Hz, 3H).HRMS(ESI) calcd for C₁₉H₃₁N₂O₁₂S₃[M+H]⁺ m/z 575.1020: found 575.1034.

5-(Bis(2-((methylsulfonyl)oxy)ethyl)amino)-4-(ethylsulfonyl)-2-nitrobenzoicacid 119

tert-Butyl5-(bis(2-((methylsulfonyl)oxy)ethyl)amino)-4-(ethylsulfonyl)-2-nitrobenzoate118 (14.80, 25.76) in CH₂Cl₂ (80 mL) was treated with TFA (40 mL) at 5°C. The reaction was stirred at the room temperature for 2 h, and thesolvents were removed under reduced pressure. The residue was thendissolved in EtOAc and the solvent was evaporated to dryness to removethe excess TFA. The yellow residue was dissolved in CH₂Cl₂ andprecipitated with iPr₂O to give5-(bis(2-((methylsulfonyl)oxy)ethyl)amino)-4-(ethylsulfonyl)-2-nitrobenzoicacid 119 (13.22 g, 99%) as a yellow gum. ¹HNMR [(CD₃)₂SO] δ 8.50 (s,1H), 7.88 (s, 1H), 4.35 (t, J=5.0 Hz, 4H), 3.75 (t, J=5.0 Hz, 4H), 3.65(q, J=7.3 Hz, 2H), 3.15 (s, 6H), 1.07 (t, J=7.4 Hz, 3H). HRMS(ESI) calcdfor C₁₅H₂₃N₂O₁₂S₃[M+H]⁺ m/z 519.0413: found 519.0408.

((5-((2-Hydroxyethyl)(methyl)carbamoyl)-2-(ethylsulfonyl)-4-nitrophenyl)azanediyl)bis(ethane-2,1-diyl)dimethanesulfonate (120)

A solution of5-(bis(2-((methylsulfonyl)oxy)ethyl)amino)-4-(ethylsulfonyl)-2-nitrobenzoicacid 119 (3.21 g, 6.19 mmol) in CH₂Cl₂ (50 mL) and CH₃CN (5 mL) wastreated with MgO (4.99 g, 123.81 mmol) at the room temperature thencooled to 0° C. and treated with oxalyl chloride (3.19 mL, 37.14 mmol)and DMF (3 drops). The reaction mixture was stirred at 0° C. for 1 hthen warmed to the room temperature for 3 h. The mixture was filteredthrough a short pad of Celite and the solvents were removed underreduced pressure. The residue was dissolved in CH₂Cl₂ (105 mL) and THF(25 mL), cooled to 0° C. and treated with 2-(methylamino)ethanol (1.66mL, 20.64 mmol) and warm to the room temperature for 20 min. The mixturewas washed with water (3×), dried with Na₂SO₄ and concentrated underreduced pressure. The residues was purified by flash columnchromatography on silica gel eluting with CH₂Cl₂/MeOH (19:1) to give((5-((2-hydroxyethyl)(methyl)carbamoyl)-2-(ethylsulfonyl)-4-nitrophenyl)azanediyl)bis(ethane-2,1-diyl)dimethanesulfonate 120 (2.35 g, 66%) as a mixture of atropisomers, as ayellow gum. ¹HNMR [(CD₃)₂SO] δ 8.64 (s, 0.3H), 8.63 (s, 0.7H), 7.71 (s,0.4H), 7.65 (s, 0.6H), 4.83-4.78 (2t, J=5.2 Hz, 1H), 4.36-4.33 (m, 4H),3.78-3.75 (m, 4H), 3.69-3.53 (m, 6H), 3.15 (s, 6H), 3.04 (s, 1.6H), 2.86(s, 1.4H), 1.10 (t, J=7.4 Hz, 3H). HRMS(ESI) calcd forC₁₈H₃₀N₃O₁₂S₃[M+H]⁺ m/z 576.0972: found 576.0986.

5-(Bis(2-bromoethyl)amino)-N-(2-hydroxyethyl)-N-methyl-4-(ethylsulfonyl)-2-nitrobenzamide(18)

((5-((2-Hydroxyethyl)(methyl)carbamoyl)-2-(ethylsulfonyl)-4-nitrophenyl)azanediyl)bis(ethane-2,1-diyl)dimethanesulfonate 120 (2.00 g, 3.47 mmol) was dissolved in acetone (50mL) and treated with LiBr (6.03 g, 69.49 mmol) at the room temperature.The reaction mixture was stirred overnight and the solvent was removed.The residue was dissolved in EtOAc and washed with water (2×), driedwith Na₂SO₄ and concentrated under reduced pressure. The crude productwas purified by flash column chromatography on silica gel eluting withCH₂Cl₂/MeOH (20:1) to give5-(bis(2-bromoethyl)amino)-N-(2-hydroxyethyl)-N-methyl-4-(ethylsulfonyl)-2-nitrobenzamide18 (1.81 g, 96%) as a mixture of atropisomers, as a yellow gum. ¹HNMR[(CD₃)₂SO] δ 8.64 (s, 0.4H), 8.63 (s, 0.6H), 7.70 (s, 0.6H), 7.66 (s,0.4H), 4.83-4.77 (2t, J=5.5 Hz, 1H), 3.84-3.76 (m, 4H), 3.73-3.66 (m,3H), 3.64-3.58 (m, 4H), 3.55-3.52 (m, 1H), 3.23-3.07 (m, 2H), 3.04 (s,1.6H), 2.86 (s, 1.4H), 1.10 (t, J=7.4 Hz, 3H). HRMS(ESI) calcd forC₁₆H₂₃Br₂N₃NaO₆S [M+Na]⁺ m/z 565.9556: found 565.9567.

5-(Bis(2-bromoethyl)amino)-N-(2-hydroxyethyl)-4-(methylsulfonyl)-2-nitrobenzamide(301)

Method 1

5-(Bis(2-chloroethyl)amino)-N-(2-hydroxyethyl)-4-(methylsulfonyl)-2-nitrobenzamide(303)

A stirred solution of5-(bis(2-hydroxyethyl)amino)-4-(methylsulfonyl)-2-nitrobenzoic acid 104(490 mg, 1.41 mmol) in SOCl₂ (12.5 mL) and DMF (3 drops) was heatedunder reflux for 4 h. The excess SOCl₂ was removed by distillation underreduced pressure and the residue dissolved in CH₂Cl₂ (5 mL) and THF (3mL), cooled to 0° C. and treated with 2-aminoethanol (296 μL, 4.91mmol). The reaction mixture was stirred at 0° C. for 20 min then warmedto the room temperature, acidified with aqueous HCl (0.5 M, 4 mL) andextracted with EtOAc (2×). The combined organic phases were washed withbrine, dried with Na₂SO₄ and evaporated to dryness under reducepressure. The crude product was purified by flash column chromatographyon silica gel eluting with CH₂Cl₂/MeOH (25:1) to give5-(bis(2-chloroethyl)amino)-N-(2-hydroxyethyl)-4-(methylsulfonyl)-2-nitrobenzamide303 (300 mg, 50%) as a yellow gum. ¹HNMR [(CD₃)₂SO] δ 8.80 (t, J=5.7 Hz,1H), 8.51 (s, 1H), 7.69 (s, 1H), 4.79 (t, J=5.4 Hz, 1H), 3.81-3.77 (m,4H), 3.72-3.69 (m, 4H), 3.55-3.51 (m, 2H), 3.48 (s, 3H), 3.34-3.29 (m,2H). LRMS (APCI) calcd for C₁₄H₂₀Cl₂N₃O₆S [M+H]⁺ m/z 429.30: found429.00.

5-(Bis(2-bromoethyl)amino)-N-(2-hydroxyethyl)-4-(methylsulfonyl)-2-nitrobenzamide(301)

A solution of5-(bis(2-chloroethyl)amino)-N-(2-hydroxyethyl)-4-(methylsulfonyl)-2-nitrobenzamide 303 (250 mg, 0.58 mmol) in 3-methyl-2-butanone (10 mL) wastreated with LiBr (1.02 g, 11.75 mmol) and heated to reflux overnight.The reaction mixture was cooled to the room temperature and the solventwas removed under reduced pressure. The residue was dissolved in EtOAcand washed with water (3×), dried with Na₂SO₄ and concentrated underreduced pressure. The crude mixture was resubmitted to LiBr (2×) andworked up as above. The final product was purified by flash columnchromatography on silica gel eluting with CH₂Cl₂/MeOH (20:1) to give5-(bis(2-bromoethyl)amino)-N-(2-hydroxyethyl)-4-(methylsulfonyl)-2-nitrobenzamide301 (250 mg, 83%) as a pale yellow solid. M.p. and ¹HNMR identical tothat previously reported (Denny et al, WO2005/042471A1).

5-(Bis(2-bromoethyl)amino)-N-(2-hydroxyethyl)-4-(methylsulfonyl)-2-nitrobenzamide(301)

Method 2

((5-((2-Hydroxyethyl)carbamoyl)-2-(methylsulfonyl)-4-nitrophenyl)azanediyl)bis(ethane-2,1-diyl)dimethanesulfonate (304)

A solution of5-(bis(2-((methylsulfonyl)oxy)ethyl)amino)-4-(methylsulfonyl)-2-nitrobenzoicacid 109 (3.35 g, 6.64 mmol) in CH₂Cl₂ (100 mL) and CH₃CN (25 mL) wastreated with MgO (4.01 g, 99.60 mmol) at the room temperature thencooled to 0° C. and treated with oxalyl chloride (3.42 mL, 39.84 mmol)and DMF (3 drops). The reaction mixture was stirred at 0° C. for 1 hthen warmed to the room temperature for 3 h. The mixture was filteredthrough a short pad of Celite and the solvents were removed underreduced pressure. The residue was dissolved in CH₂Cl₂ (100 mL) and THF(25 mL), cooled to 0° C. and treated with ethanol amine (601 μL, 9.96mmol) and warm to the room temperature for 20 min. The mixture waswashed with water (3×), dried with Na₂SO₄ and concentrated under reducedpressure. The residues was purified by flash column chromatography onsilica gel eluting with CH₂Cl₂/MeOH (19:1) to give((5-((2-hydroxyethyl)carbamoyl)-2-(methylsulfonyl)-4-nitrophenyl)azanediyl)bis(ethane-2,1-diyl)dimethanesulfonate 304 (3.40 g, 94%) as a yellow gum.

¹HNMR [(CD₃)₂SO] δ 8.71 (t, J=5.7 Hz, 1H), 8.51 (s, 1H), 7.73 (s, 1H),4.77 (t, J=5.0 Hz, 1H), 4.35 (t, J=5.2 Hz, 4H), 3.72 (t, J=5.2 Hz, 4H),3.55-3.51 (m, 2H), 3.44 (s, 3H), 3.34-3.29 (m, 2H), 3.16 (s, 6H).HRMS(ESI) calcd for C₁₆H₂₅N₃NaO₁₂S₃[M+Na]⁺ m/z 570.0497: found 570.0493.

5-(Bis(2-bromoethyl)amino)-N-(2-hydroxyethyl)-4-(methylsulfonyl)-2-nitrobenzamide(301)

((5-((2-Hydroxyethyl)carbamoyl)-2-(methylsulfonyl)-4-nitrophenyl)azanediyl)bis(ethane-2,1-diyl)dimethanesulfonate 304 (3.40 g, 6.21 mmol) was dissolved in acetone (180mL) and treated with LiBr (10.78 g, 124.18 mmol) at the roomtemperature. The reaction mixture was stirred overnight and the solventwas removed. The residue was dissolved in EtOAc and washed with water(2×), dried with Na₂SO₄ and concentrated under reduced pressure. Thecrude product was purified by flash column chromatography on silica geleluting with CH₂Cl₂/MeOH (20:1) and further recrystallized fromCH₂Cl₂/MeOH (4:1) and iPr₂O to give5-(bis(2-bromoethyl)amino)-N-(2-hydroxyethyl)-4-(methylsulfonyl)-2-nitrobenzamide28 (3.06 g, 95%) as a pale yellow solid. M.p. and ¹HNMR identical tothat previously reported (Denny et al, WO2005/042471A1).

2-(5-(bis(2-bromoethyl)amino)-N-methyl-4-(methylsulfonyl)-2-nitrobenzamido)ethyldihydrogen phosphate (10)2-(5-(Bis(2-bromoethyl)amino)-N-methyl-4-(methylsulfonyl)-2-nitrobenzamido)ethyldi-tert-butyl phosphate (122)

A solution of5-(Bis(2-bromoethyl)amino)-N-(2-hydroxyethyl)-N-methyl-4-(methylsulfonyl)-2-nitrobenzamide14 (3.13 g, 5.89 mmol) in DMF (4.2 mL) and 1H-tetrazole solution (3%,1.90 g, 27.10 mmol) in CH₃CN was treated withdi-tert-butyl-N,N-diisopropylphosphoramidite (7.44 mL, 23.56 mmol) at 5°C. The reaction mixture was stirred for 4 h at the room temperature,diluted with CH₂Cl₂ (25 mL), cooled to 0° C. and solid m-CPBA (70%, 7.78g, 44.18 mmol) added portionwise. The mixture was warmed to the roomtemperature, stirred for further 1 h and the solvents were removed underreduced pressure. The residue was dissolved in EtOAc, washed with 10%solution of sodium disulfite (2×) and 5% solution of sodium bicarbonate(3×), dried with Na₂SO₄ and concentrated under reduced pressure. Thecrude product was purified by flash column chromatography on silica geleluting with CH₂Cl₂/MeOH (25:1) to give2-(5-(bis(2-bromoethyl)amino)-N-methyl-4-(methylsulfonyl)-2-nitrobenzamido)ethyldi-tert-butyl phosphate 122 (3.23 g, 76%) as a mixture of atropisomers,as a yellow gum. ¹HNMR [(CD₃)₂SO] δ 8.67 (s, 0.5H), 8.66 (s, 0.5H), 7.78(s, 0.5H), 7.60 (s, 0.5H), 4.15-4.02 (m, 2H), 3.85-3.81 (m, 4H),3.78-3.66 (m, 2H), 3.64-3.61 (m, 4H), 3.49 (s, 3H), 3.07 (s, 1.5H), 2.89(s, 1.5H), 1.44 (s, 10H), 1.40 (s, 8H). HRMS(ESI) calcd forC₂₃H₃₈Br₂N₃NaO₉PS [M+Na]⁺ m/z 744.0301: found 744.0325.

2-(5-(Bis(2-bromoethyl)amino)-N-methyl-4-(methylsulfonyl)-2-nitrobenzamido)ethyldihydrogen phosphate (10)

2-(5-(Bis(2-bromoethyl)amino)-N-methyl-4-(methylsulfonyl)-2-nitrobenzamido)ethyldi-tert-butyl phosphate 122 (3.23 g, 4.46 mmol) in CH₂Cl₂ (17 mL) wascooled to 5° C. and treated with TFA (17 mL). The reaction mixture wasstirred for 1 h at the room temperature, and the solvents were removedunder reduced pressure. The residue was triturated with CH₂Cl₂/iPr₂Othen dissolved in CH₃CN. The solvent was removed under reduced pressure(water bath 29° C.) to provide2-(5-(bis(2-bromoethyl)amino)-N-methyl-4-(methylsulfonyl)-2-nitrobenzamido)ethyldihydrogen phosphate 10 (2.72 g, 100%) as a mixture of atropisomers, asa yellow gum. ¹HNMR [(CD₃)₂SO] δ 8.66 (s, 0.5H), 8.65 (s, 0.5H), 7.78(s, 0.5H), 7.63 (s, 0.5H), 4.11-4.06 (m, 2H), 3.84-3.81 (m, 4H),3.78-3.65 (m, 2H), 3.61-3.58 (m, 4H), 3.46 (s, 3H), 3.04 (s, 1.5H), 2.86(s, 1.5H). HRMS(ESI) calcd for C₁₅H₂₂Br₂N₃NaO₉PS [M+Na]⁺ m/z 631.9065:found 631.9073.

2-(5-(bis(2-bromoethyl)amino)-4-(ethylsulfonyl)-N-methyl-2-nitrobenzamido)ethyldihydrogen phosphate (11)2-(5-(Bis(2-bromoethyl)amino)-N-methyl-4-(ethylsulfonyl)-2-nitrobenzamido)ethyldi-tert-butyl phosphate (123)

A solution of5-(bis(2-bromoethyl)amino)-N-(2-hydroxyethyl)-N-methyl-4-(ethylsulfonyl)-2-nitrobenzamide18 (1.80 g, 3.30 mmol) in DMF (2.0 mL) and 1H-tetrazole solution (3%,1.06 g, 15.18 mmol) in CH₃CN was treated withdi-tert-butyl-N,N-diisopropylphosphoramidite (4.16 mL, 13.20 mmol) at 5°C. The reaction mixture was stirred for 4 h at the room temperature,diluted with CH₂Cl₂ (15 mL), cooled to 0° C. and solid m-CPBA (70%, 4.36g, 24.75 mmol) added portionwise. The mixture was warmed to the roomtemperature, stirred for further 1 h and the solvents were removed underreduced pressure. The residue was dissolved in EtOAc, washed with 10%solution of sodium disulfite (2×) and 5% solution of sodium bicarbonate(3×), dried with Na₂SO₄ and concentrated under reduced pressure. Thecrude product was purified by flash column chromatography on silica geleluting with CH₂Cl₂/MeOH (25:1) to give2-(5-(bis(2-bromoethyl)amino)-N-methyl-4-(ethylsulfonyl)-2-nitrobenzamido)ethyldi-tert-butyl phosphate 123 (2.16 g, 89%) as a mixture of atropisomers,as a yellow gum. ¹HNMR [(CD₃)₂SO] δ 8.65 (s, 0.5H), 8.64 (s, 0.5H), 7.77(s, 0.5H), 7.59 (s, 0.5H), 4.14-4.11 (m, 2H), 3.84-3.81 (m, 5H),3.74-3.66 (m, 3H), 3.63-3.60 (m, 4H), 3.07 (s, 1.5H), 2.89 (s, 1.5H),1.44 (s, 10H), 1.40 (s, 8H), 1.10 (t, J=7.3 Hz, 3H). HRMS(ESI) calcd forC₂₄H₄₀Br₂N₃NaO₉PS [M+Na]⁺ m/z 758.0469: found 758.0440.

2-(5-(bis(2-bromoethyl)amino)-4-(ethylsulfonyl)-N-methyl-2-nitrobenzamido)ethyldihydrogen phosphate (11)

2-(5-(Bis(2-bromoethyl)amino)-N-methyl-4-(ethylsulfonyl)-2-nitrobenzamido)ethyldi-tert-butyl phosphate 123 (2.16 g, 2.93 mmol) in CH₂Cl₂ (25 mL) wascooled to 5° C. and treated with TFA (5 mL). The reaction mixture wasstirred for 1 h at the room temperature, and the solvents were removedunder reduced pressure (water bath 29° C.). The gum was then trituratedwith iPr₂O to provide2-(5-(bis(2-bromoethyl)amino)-4-(ethylsulfonyl)-N-methyl-2-nitrobenzamido)ethyldihydrogen phosphate 11 (1.59 g, 87%) as a mixture of atropisomers, as ayellow gum. ¹HNMR [(CD₃)₂SO] δ 8.65 (s, 0.6H), 8.64 (s, 0.4H), 7.76 (s,0.5H), 7.62 (s, 0.5H), 4.10-3.99 (m, 2H), 3.84-3.80 (m, 4H), 3.74-3.68(m, 3H), 3.63-3.57 (m, 5H), 3.06 (s, 1.4H), 2.89 (s, 1.6H), 1.12-1.08(m, 3H). Anal. calcd for C₁₆H₂₄Br₂N₃O₉PS. (0.25iPr₂O+0.1CH₂Cl₂): C,32.10; H, 4.16; N, 6.38; P, 4.70%; found: C, 32.36; H, 4.09; N, 6.13; P,4.34%.

2-(5-(Bis(2-bromoethyl)amino)-4-(methylsulfonyl)-2-nitrobenzamido)ethyldihydrogen phosphate (300)2-(5-(Bis(2-bromoethyl)amino)-4-(methylsulfonyl)-2-nitrobenzamido)ethyldi-tert-butyl phosphate (302)

A solution of5-(bis(2-bromoethyl)amino)-N-(2-hydroxyethyl)-4-(methylsulfonyl)-2-nitrobenzamide301 (3.00 g, 5.80 mmol) in DMF (4.1 mL) and 1H-tetrazole solution (3%,1.87 g, 26.68 mmol) in CH₃CN was treated withdi-tert-butyl-N,N-diisopropylphosphoramidite (7.32 mL, 23.20 mmol) at 5°C. The reaction mixture was stirred for 4 h at the room temperature,diluted with CH₂Cl₂ (25 mL), cooled to 0° C. and solid m-CPBA (70%,10.22 g, 58.00 mmol) added portionwise. The mixture was warmed to theroom temperature, stirred for further 1 h and the solvents were removedunder reduced pressure. The residue was dissolved in EtOAc, washed with10% solution of sodium disulfite (2×) and 5% solution of sodiumbicarbonate (3×), dried with Na₂SO₄ and concentrated under reducedpressure. The crude product was purified by flash column chromatographyon silica gel eluting with CH₂Cl₂/MeOH (25:1) to give2-(5-(bis(2-bromoethyl)amino)-4-(methylsulfonyl)-2-nitrobenzamido)ethyldi-tert-butyl phosphate 302 (2.78 g, 68%) as a yellow gum. ¹HNMR[(CD₃)₂SO] δ 8.94 (t, J=5.6 Hz, 1H), 8.53 (s, 1H), 7.73 (s, 1H),4.00-3.96 (m, 2H), 3.77-3.74 (m, 4H), 3.64-3.61 (m, 4H), 3.52-3.48 (m,2H), 3.50 (s, 3H), 1.43 (s, 18H). HRMS(ESI) calcd for C₂₂H₃₆Br₂N₃NaO₉PS[M+Na]⁺ m/z 730.0163: found 730.0169.

2-(5-(Bis(2-bromoethyl)amino)-4-(methylsulfonyl)-2-nitrobenzamido)ethyldihydrogen phosphate (300)

2-(5-(Bis(2-bromoethyl)amino)-4-(methylsulfonyl)-2-nitrobenzamido)ethyldi-tert-butyl phosphate 302 (2.70 g, 3.81 mmol) in CH₂Cl₂ (14 mL) wascooled to 5° C. and treated with TFA (14 mL). The reaction mixture wasstirred for 1 h at the room temperature, and the solvents were removedunder reduced pressure. The residue was triturated with CH₂Cl₂/iPr₂Othen dissolved in CH₃CN. The solvent was removed under reduced pressure(water bath 29° C.) to provide2-(5-(bis(2-bromoethyl)amino)-4-(methylsulfonyl)-2-nitrobenzamido)ethyldihydrogen phosphate 300 (2.27 g, 100%) as a yellow gum. ¹HNMR[(CD₃)₂SO] δ 8.93 (t, J=5.8 Hz, 1H), 8.52 (s, 1H), 7.76 (s, 1H),3.98-3.93 (m, 2H), 3.77-3.74 (m, 4H), 3.64-3.61 (m, 4H), 3.50-3.45 (m,2H), 3.50 (s, 3H). HRMS(ESI) calcd for C₁₄H₂₀Br₂N₃NaO₉PS [M+Na]⁺ m/z617.8899: found 617.8917.

(5-(Bis(2-bromoethyl)amino)-4-(methylsulfonyl)-2-nitrophenyl)(4-methylpiperazine-1-yl)methanone(22)((5-(4-methylpiperazine-1-carbonyl)-2-(methylsulfonyl)-4-nitrophenyl)azanediyl)bis(ethane-2,1-diyl)dimethanesulfonate(124)

A solution of5-(bis(2-((methylsulfonyl)oxy)ethyl)amino)-4-(methylsulfonyl)-2-nitrobenzoicacid 109 (770 mg, 1.53 mmol) in CH₂Cl₂ (20 mL) and CH₃CN (4 mL) wastreated with MgO (1.23 g, 30.52 mmol) at the room temperature thencooled to 0° C. and treated with oxalyl chloride (786 μL, 9.16 mmol) andDMF (3 drops). The reaction mixture was stirred at 0° C. for 1 h thenwarmed to the room temperature for 4 h. The mixture was filtered througha short pad of Celite and the solvents were removed under reducedpressure. The residue was dissolved in CH₂Cl₂ (20 mL) and THF (20 mL),cooled to 0° C. and treated with 1-methylpiperazine (459 mg, 4.58 mmol)and warm to the room temperature for 30 min. The mixture was washed withwater (3×), dried with Na₂SO₄ and concentrated under reduced pressure.The residues was purified by flash column chromatography on silica geleluting with CH₂Cl₂/MeOH (19:1) to give((5-(4-methylpiperazine-1-carbonyl)-2-(methylsulfonyl)-4-nitrophenyl)azanediyl)bis(ethane-2,1-diyl)dimethanesulfonate124 (600 mg, 67%) as a yellow gum. ¹HNMR [(CD₃)₂SO] δ 8.65 (s, 1H), 7.68(s, 1H), 4.34 (t, J=5.1 Hz, 4H), 3.79-3.78 (m, 5H), 3.49 (br, 1H), 3.44(s, 3H), 3.24-3.17 (m, 2H), 3.14 (s, 6H), 2.33 (br, 3H) 2.20 (s, 3H),2.09 (br, 1H). HRMS(ESI) calcd for C₁₉H₃₁N₄O₁₁S₃[M+H]⁺ m/z 587.1146:found 587.1148.

(5-(bis(2-bromoethyl)amino)-4-(methylsulfonyl)-2-nitrophenyl)(4-methylpiperazine-1-yl)methanone(22)

((5-(4-Methylpiperazine-1-carbonyl)-2-(methylsulfonyl)-4-nitrophenyl)azanediyl)bis(ethane-2,1-diyl)dimethanesulfonate 124 (1.2 g, 2.05 mmol) was dissolved in acetone (40mL) and treated with LiBr (3.55 g, 40.91 mmol) at the room temperature.The reaction mixture was stirred overnight and the solvent was removed.The residue was dissolved in EtOAc and washed with water (2×), driedwith Na₂SO₄ and concentrated under reduced pressure. The crude productwas purified by flash column chromatography on silica gel eluting withCH₂Cl₂/MeOH (20:1) to give(5-(bis(2-bromoethyl)amino)-4-(methylsulfonyl)-2-nitrophenyl)(4-methylpiperazine-1-yl)methanone22 (1.01 g, 89%) as a yellow gum. ¹HNMR [(CD₃)₂SO] δ 8.65 (s, 1H), 7.69(s, 1H), 3.85-3.80 (m, 4H), 3.78-3.69 (m, 2H), 3.62 (t, J=6.8 Hz, 4H),3.58-3.51 (m, 1H), 3.48 (s, 3H), 3.23-3.17 (m, 2H), 2.46-2.29 (m, 2H),2.21 (s, 3H), 2.13 (br, 1H). HRMS(ESI) calcd for C₁₇H₂₄Br₂KN₄O₅S[M+K]+m/z 592.9466: found 592.9474.

(5-(bis(2-bromoethyl)amino)-4-(methylsulfonyl)-2-nitrophenyl)(4-ethylpiperazine-1-yl)methanone(23)(5-(bis(2-chloroethyl)amino)-4-(methylsulfonyl)-2-nitrophenyl)(4-ethylpiperazine-1-yl)methanone(131)

A stirred solution of5-(bis(2-hydroxyethyl)amino)-4-(methylsulfonyl)-2-nitrobenzoic acid 104(1.62 g, 4.65 mmol) in SOCl₂ (40 mL) and DMF (3 drops) was heated underreflux for 4 h. The excess SOCl₂ was removed by distillation underreduced pressure and the residue was dissolved in CH₂Cl₂ (32 mL) and THF(32 mL), cooled to 0° C. and treated with 1-ethylpiperazine (1.60 g,14.01 mmol) and warm to the room temperature for 30 min. The mixture waswashed with water (3×), dried with Na₂SO₄ and concentrated under reducedpressure. The residues was purified by flash column chromatography onsilica gel eluting with CH₂Cl₂/MeOH (19:1) to give(5-(bis(2-chloroethyl)amino)-4-(methylsulfonyl)-2-nitrophenyl)(4-ethylpiperazine-1-yl)methanone131 (1.12 g, 50%) as a yellow gum. ¹HNMR [(CD₃)₂SO]δ 8.65 (s, 1H), 7.68(s, 1H), 3.78-3.77 (m, 8H), 3.69 (br, 1H), 3.58 (br, 1H), 3.47 (s, 3H),3.17 (br, 2H), 2.42 (br, 1H), 2.39-2.33 (m, 3H), 2.25-1.98 (m, 1H),1.02-0.98 (t, J=7.2 Hz, 3H). HRMS(ESI) calcd for C₁₈H₂₇Cl₂N₄O₅S [M+H]⁺m/z 481.1074: found 481.1073.

(5-(bis(2-bromoethyl)amino)-4-(methylsulfonyl)-2-nitrophenyl)(4-ethylpiperazine-1-yl)methanone(23)

A solution of(5-(bis(2-chloroethyl)amino)-4-(methylsulfonyl)-2-nitrophenyl)(4-ethylpiperazine-1-yl)methanone131 (4.50 g, 9.37 mmol) in 3-methyl-2-butanone (200 ml) was treated withLiBr (16.28 g, 187.46 mmol) and heated to reflux overnight. The reactionmixture was cooled to the room temperature and the solvent was removedunder reduced pressure. The residue was dissolved in EtOAc and washedwith water (3×), dried with Na₂SO₄ and concentrated under reducedpressure. The crude mixture was resubmitted to LiBr (2×) and worked upas above. The final product was purified by flash column chromatographyon silica gel eluting with CH₂Cl₂/MeOH (20:1) to give(5-(bis(2-bromoethyl)amino)-4-(methylsulfonyl)-2-nitrophenyl)(4-ethylpiperazine-1-yl)methanone23 (1.92 g, 36%) as a yellow gum. ¹HNMR [(CD₃)₂SO] δ 8.65 (s, 1H), 7.69(s, 1H), 3.83 (q, J=7.4 Hz, 4H), 3.74-3.67 (m, 1H), 3.64-3.54 (m, 6H),3.48 (s, 3H), 3.23-3.16 (m, 2H), 2.46-2.40 (m, 1H), 2.39-2.32 (m, 3H),2.25-1.98 (m, 1H), 1.02-0.98 (t, J=7.2 Hz, 3H). HRMS(ESI) calcd forC₁₈H₂₇Br₂N₄O₅S [M+H]⁺ m/z 569.0063: found 569.0042.

(5-(bis(2-bromoethyl)amino)-4-(methylsulfonyl)-2-nitrophenyl)(4-isopropylpiperazine-1-yl)methanone(24)(5-(bis(2-chloroethyl)amino)-4-(methylsulfonyl)-2-nitrophenyl)(4-isopropylpiperazine-1-yl)methanone(132)

A stirred solution of5-(bis(2-hydroxyethyl)amino)-4-(methylsulfonyl)-2-nitrobenzoic acid 104(1.09 g, 3.13 mmol) in SOCl₂ (30 mL) and DMF (3 drops) was heated underreflux for 4 h. The excess SOCl₂ was removed by distillation underreduced pressure and the residue was dissolved in CH₂Cl₂ (20 mL) and THF(20 mL), cooled to 0° C. and treated with 1-isopropylpiperazine (1.20 g,9.39 mmol) and warm to the room temperature for 30 min. The mixture waswashed with water (3×), dried with Na₂SO₄ and concentrated under reducedpressure. The residues was purified by flash column chromatography onsilica gel eluting with CH₂Cl₂/MeOH (19:1) to give(5-(bis(2-chloroethyl)amino)-4-(methylsulfonyl)-2-nitrophenyl)(4-isopropylpiperazine-1-yl)methanone132 (1.06 g, 69%) as a yellow gum. ¹HNMR [(CD₃)2SO] δ 8.65 (s, 1H), 7.68(s, 1H), 3.78-3.77 (m, 8H), 3.68 (br, 1H), 3.56 (br, 1H), 3.47 (s, 3H),3.16 (br, 2H), 2.74-2.65 (m, 1H), 2.57 (br, 2H), 2.39 (br, 1H),2.34-2.26 (m, 1H), 0.99 (d, J=6.5 Hz, 6H). HRMS(ESI) calcd forC₁₉H₂₉Cl₂N₄O₅S [M+H]⁺ m/z 495.1230: found 495.1217.

(5-(bis(2-bromoethyl)amino)-4-(methylsulfonyl)-2-nitrophenyl)(4-isopropylpiperazine-1-yl)methanone(24)

A solution of(5-(bis(2-chloroethyl)amino)-4-(methylsulfonyl)-2-nitrophenyl)(4-isopropylpiperazine-1-yl)methanone132 (1.06 g, 2.15 mmol) in 3-methyl-2-butanone (100 ml) was treated withLiBr (3.73 g, 42.90 mmol) and heated to reflux overnight. The reactionmixture was cooled to the room temperature and the solvent was removedunder reduced pressure. The residue was dissolved in EtOAc and washedwith water (3×), dried with Na₂SO₄ and concentrated under reducedpressure. The crude mixture was resubmitted to LiBr (2×) and worked upas above. The final product was purified by flash column chromatographyon silica gel eluting with CH₂Cl₂/MeOH (20:1) to give(5-(bis(2-bromoethyl)amino)-4-(methylsulfonyl)-2-nitrophenyl)(4-isopropylpiperazine-1-yl)methanone24 (660 mg, 53%) as a yellow gum. ¹HNMR [(CD₃)₂SO] δ 8.65 (s, 1H), 7.69(s, 1H), 3.83 (q, J=7.0 Hz, 4H), 3.67-3.53 (m, 6H), 3.50 (s, 3H), 3.18(br, 2H), 2.73-2.67 (m, 1H), 2.60-2.52 (m, 1H), 2.47-2.40 (m, 1H),2.33-2.23 (m, 1H), 2.15-2.06 (m, 1H), 0.97 (d, J=6.5 Hz, 6H). HRMS(ESI)calcd for C₁₉H₂₉Br₂N₄O₅S [M+H]⁺ m/z 583.0220: found 583.0203.

(5-(bis(2-bromoethyl)amino)-4-(ethylsulfonyl)-2-nitrophenyl)(4-methylpiperazine-1-yl)methanone(25)((5-(4-methylpiperazine-1-carbonyl)-2-(ethylsulfonyl)-4-nitrophenyl)azanediyl)bis(ethane-2,1-diyl)dimethanesulfonate(133)

A solution of5-(bis(2-((methylsulfonyl)oxy)ethyl)amino)-4-(ethylsulfonyl)-2-nitrobenzoicacid 119 (2.33 g, 4.49 mmol) in CH₂Cl₂ (60 mL) and CH₃CN (12 mL) wastreated with MgO (3.59 g, 89.87 mmol) at the room temperature thencooled to 0° C. and treated with oxalyl chloride (2.31 mL, 26.96 mmol)and DMF (3 drops). The reaction mixture was stirred at 0° C. for 1 hthen warmed to the room temperature for 4 h. The mixture was filteredthrough a short pad of Celite and the solvents were removed underreduced pressure. The residue was dissolved in CH₂Cl₂ (60 mL) and THF(60 mL), cooled to 0° C. and treated with 1-methylpiperazine (1.35 g,13.48 mmol) and warm to the room temperature for 30 min. The mixture waswashed with water (3×), dried with Na₂SO₄ and concentrated under reducedpressure. The residues was purified by flash column chromatography onsilica gel eluting with CH₂Cl₂/MeOH (19:1) to give((5-(4-methylpiperazine-1-carbonyl)-2-(ethylsulfonyl)-4-nitrophenyl)azanediyl)bis(ethane-2,1-diyl)dimethanesulfonate133 (1.78 g, 66%) as a yellow gum. ¹HNMR [(CD₃)₂SO] δ 8.63 (s, 1H), 7.67(s, 1H), 4.36-4.33 (m, 4H), 3.80-3.76 (m, 6H), 3.66-3.60 (m, 3H), 3.48(br, 2H), 3.22-3.17 (m, 2H), 3.15 (s, 6H), 2.20 (s, 3H), 2.13 (br, 1H),1.10 (t, J=7.4 Hz, 3H). HRMS(ESI) calcd for C₂₀H₃₃N₄O₁₁S₃[M+H]⁺ m/z601.1302: found 601.1299.

(5-(bis(2-bromoethyl)amino)-4-(ethylsulfonyl)-2-nitrophenyl)(4-methylpiperazine-1-yl)methanone(25)

((5-(4-Methylpiperazine-1-carbonyl)-2-(ethylsulfonyl)-4-nitrophenyl)azanediyl)bis(ethane-2,1-diyl)dimethanesulfonate133 (1.78 g, 2.96 mmol) was dissolved in acetone (70 mL) and treatedwith LiBr (5.15 g, 59.27 mmol) at the room temperature. The reactionmixture was stirred overnight and the solvent was removed. The residuewas dissolved in EtOAc and washed with water (2×), dried with Na₂SO₄ andconcentrated under reduced pressure. The crude product was purified byflash column chromatography on silica gel eluting with CH₂Cl₂/MeOH(20:1) to give(5-(bis(2-bromoethyl)amino)-4-(ethylsulfonyl)-2-nitrophenyl)(4-methylpiperazine-1-yl)methanone25 (1.44 g, 85%) as a yellow gum. ¹HNMR [(CD₃)₂SO] δ 8.63 (s, 1H), 7.68(s, 1H), 3.85-3.80 (m, 4H), 3.72-3.66 (m, 3H), 3.63-3.59 (m, 5H), 3.19(br, 2H), 2.46-2.29 (m, 3H), 2.21 (s, 3H), 2.17 (br, 1H), 1.11 (t, J=7.4Hz, 3H). HRMS(ESI) calcd for C₁₈H₂₆Br₂KN₄O₅S [M+K]⁺ m/z 606.9622: found606.9615.

(5-(bis(2-bromoethyl)amino)-4-(ethylsulfonyl)-2-nitrophenyl)(4-ethylpiperazine-1-yl)methanone(26)((5-(4-ethylpiperazine-1-carbonyl)-2-(ethylsulfonyl)-4-nitrophenyl)azanediyl)bis(ethane-2,1-diyl)dimethanesulfonate(134)

A solution of5-(bis(2-((methylsulfonyl)oxy)ethyl)amino)-4-(ethylsulfonyl)-2-nitrobenzoicacid 119 (1.96 g, 3.78 mmol) in CH₂Cl₂ (50 mL) and CH₃CN (10 mL) wastreated with MgO (3.02 g, 75.60 mmol) at the room temperature thencooled to 0° C. and treated with oxalyl chloride (1.95 mL, 22.68 mmol)and DMF (3 drops). The reaction mixture was stirred at 0° C. for 1 hthen warmed to the room temperature for 4 h. The mixture was filteredthrough a short pad of Celite and the solvents were removed underreduced pressure. The residue was dissolved in CH₂Cl₂ (50 mL) and THF(50 mL), cooled to 0° C. and treated with 1-ethylpiperazine (1.29 g,11.34 mmol) and warm to the room temperature for 30 min. The mixture waswashed with water (3×), dried with Na₂SO₄ and concentrated under reducedpressure. The residues was purified by flash column chromatography onsilica gel eluting with CH₂Cl₂/MeOH (19:1) to give((5-(4-ethylpiperazine-1-carbonyl)-2-(ethylsulfonyl)-4-nitrophenyl)azanediyl)bis(ethane-2,1-diyl)dimethanesulfonate134 (1.14 g, 49%) as a yellow gum. ¹HNMR [(CD₃)₂SO] δ 8.63 (s, 1H), 7.67(s, 1H), 4.34-4.33 (m, 4H), 3.80-3.76 (m, 5H), 3.66-3.60 (m, 2H), 3.49(br, 2H), 3.22-3.17 (m, 2H), 3.15 (s, 6H), 2.39-2.32 (m, 4H), 2.18 (br,1H), 1.09 (t, J=7.4 Hz, 3H), 1.00 (t, J=7.2 Hz, 3H). HRMS(ESI) calcd forC₂₁H₃₅N₄O₁₁S₃[M+H]⁺ m/z 615.1459: found 615.1464.

(5-(bis(2-bromoethyl)amino)-4-(ethylsulfonyl)-2-nitrophenyl)(4-ethylpiperazine-1-yl)methanone(26)

((5-(4-Ethylpiperazine-1-carbonyl)-2-(ethylsulfonyl)-4-nitrophenyl)azanediyl)bis(ethane-2,1-diyl)dimethanesulfonate134 (1.14 g, 1.85 mmol) was dissolved in acetone (45 mL) and treatedwith LiBr (5.15 g, 37.09 mmol) at the room temperature. The reactionmixture was stirred overnight and the solvent was removed. The residuewas dissolved in EtOAc and washed with water (2×), dried with Na₂SO₄ andconcentrated under reduced pressure. The crude product was purified byflash column chromatography on silica gel eluting with CH₂Cl₂/MeOH(20:1) to give(5-(bis(2-bromoethyl)amino)-4-(ethylsulfonyl)-2-nitrophenyl)(4-ethylpiperazine-1-yl)methanone26 (915 mg, 85%) as a yellow gum. ¹HNMR [(CD₃)₂SO] δ 8.63 (s, 1H), 7.68(s, 1H), 3.84-3.80 (m, 4H), 3.77-3.66 (m, 4H), 3.63-3.59 (m, 5H), 3.19(br, 2H), 2.42 (br, 1H), 2.39-2.32 (m, 3H), 2.21 (br, 1H), 1.11 (t,J=7.4 Hz, 3H), 1.00 (t, J=7.2 Hz, 3H). HRMS(ESI) calcd forC₁₉H₂₉Br₂N₄O₅S [M+H]⁺ m/z 583.0220: found 583.0221.

(5-(bis(2-bromoethyl)amino)-4-(ethylsulfonyl)-2-nitrophenyl)(4-isopropylpiperazine-1-yl)methanone(27)((5-(4-isopropylpiperazine-1-carbonyl)-2-(ethylsulfonyl)-4-nitrophenyl)azanediyl)bis(ethane-2,1-diyl)dimethanesulfonate (135)

A solution of5-(bis(2-((methylsulfonyl)oxy)ethyl)amino)-4-(ethylsulfonyl)-2-nitrobenzoicacid 119 (2.00 g, 3.85 mmol) in CH₂Cl₂ (50 mL) and CH₃CN (12 mL) wastreated with MgO (3.08 g, 77.14 mmol) at the room temperature thencooled to 0° C. and treated with oxalyl chloride (1.99 mL, 23.14 mmol)and DMF (3 drops). The reaction mixture was stirred at 0° C. for 1 hthen warmed to the room temperature for 4 h. The mixture was filteredthrough a short pad of Celite and the solvents were removed underreduced pressure. The residue was dissolved in CH₂Cl₂ (50 mL) and THF(50 mL), cooled to 0° C. and treated with 1-isopropylpiperazine (1.48 g,11.57 mmol) and warm to the room temperature for 30 min. The mixture waswashed with water (3×), dried with Na₂SO₄ and concentrated under reducedpressure. The residues was purified by flash column chromatography onsilica gel eluting with CH₂Cl₂/MeOH (19:1) to give((5-(4-isopropylpiperazine-1-carbonyl)-2-(ethylsulfonyl)-4-nitrophenyl)azanediyl)bis(ethane-2,1-diyl)dimethanesulfonate135 (1.21 g, 49%) as a yellow gum. ¹HNMR [(CD₃)₂SO] δ 8.63 (s, 1H), 7.67(s, 1H), 4.34-4.33 (m, 4H), 3.78-3.77 (m, 5H), 3.66-3.60 (m, 2H),3.53-3.45 (m, 1H), 3.20 (br, 2H), 3.16 (s, 6H), 2.73-2.66 (m, 1H), 2.58(br, 1H), 2.45-2.25 (m, 3H), 1.09 (t, J=7.4 Hz, 3H), 0.97 (d, J=6.5 Hz,6H). HRMS(ESI) calcd for C₂₂H₃₇N₄O₁₁S₃[M+H]⁺ m/z 629.1615: found629.1612.

(5-(bis(2-bromoethyl)amino)-4-(ethylsulfonyl)-2-nitrophenyl)(4-isopropylpiperazine-1-yl)methanone(27)

((5-(4-Isopropylpiperazine-1-carbonyl)-2-(ethylsulfonyl)-4-nitrophenyl)azanediyl)bis(ethane-2,1-diyl)dimethanesulfonate 135 (1.16 g, 2.58 mmol) was dissolved inacetone (45 mL) and treated with LiBr (3.06 g, 35.22 mmol) at the roomtemperature. The reaction mixture was stirred overnight and the solventwas removed. The residue was dissolved in EtOAc and washed with water(2×), dried with Na₂SO₄ and concentrated under reduced pressure. Thecrude product was purified by flash column chromatography on silica geleluting with CH₂Cl₂/MeOH (20:1) to give(5-(bis(2-bromoethyl)amino)-4-(ethylsulfonyl)-2-nitrophenyl)(4-isopropylpiperazine-1-yl)methanone27 (934 mg, 85%) as a yellow gum. ¹HNMR [(CD₃)₂SO] δ 8.63 (s, 1H), 7.68(s, 1H), 3.84-3.81 (m, 4H), 3.77-3.66 (m, 3H), 3.63-3.53 (m, 5H), 3.18(br, 2H), 2.73-2.67 (m, 1H), 2.57 (br, 2H), 2.40 (br, 1H), 2.31 (br,1H), 1.10 (t, J=7.4 Hz, 3H), 0.97 (d, J=6.5 Hz, 6H). HRMS(ESI) calcd forC₂₀H₃₁Br₂N₄O₅S [M+H]⁺ m/z 597.0376: found 597.0394.

2-((2-bromoethyl)(2-((2-hydroxyethyl)carbamoyl)-4-nitrophenyl)amino)ethylmethanesulfonate (64)2-fluoro-5-nitro-N-(2-((tetrahydro-2H-pyran-2-yl)oxy)ethyl) benzamide(126)

Method 1

A stirred solution of 2-fluoro-5-nitrobenzoic acid 125 (4.56 g, 24.63mmol) in SOCl₂ (50 mL) and DMF (3 drops) was heated under reflux for 4h. The excess SOCl₂ was removed by distillation under reduced pressureand the residue was dissolved in THF (30 mL), cooled to −10° C. andtreated with 2-((tetrahydro-2H-pyran-2-yl)oxy)ethanamine (3.93 g, 27.10mmol) and warm to the room temperature for 30 min. The solvent wasevaporated and the residue was dissolved in EtOAc, washed with water(3×) and brine, dried with Na₂SO₄ and concentrated under reducedpressure. The crude product was purified by flash column chromatographyon silica gel eluting with EtOAc/Hexane (1:1) to give2-fluoro-5-nitro-N-(2-((tetrahydro-2H-pyran-2-yl)oxy)ethyl) benzamide126 (3.17 g, 41%) as a colorless oil. ¹HNMR [(CD₃)₂SO] δ 8.67 (br, 1H),8.42-8.38 (m, 2H), 7.64-7.59 (m, 1H), 4.62 (t, J=3.5 Hz, 1H), 3.55-3.41(m, 5H), 1.79-1.70 (m, 1H), 1.66-1.60 (m, 1H), 1.53-1.41 (m, 5H).HRMS(ESI) calcd for C₁₄H₁₇FN₂NaO₅ [M+Na]⁺ m/z 335.1014: found 335.1014.

Method 2

A stirred solution of 2-fluoro-5-nitrobenzoic acid 125 (15.0 g, 81.08mmol) in SOCl₂ (160 mL) and DMF (3 drops) was heated under reflux for 4h. The excess SOCl₂ was removed by distillation under reduced pressureand the residue was dissolved in THF (100 mL), cooled to −10° C. andtreated with ethanolamine (8.53 mL, 141.81 mmol) and warm to the roomtemperature for 30 min. The solvent was evaporated and the residue wasdissolved in EtOAc, washed with water (3×) and brine, dried with Na₂SO₄and concentrated under reduced pressure. The crude product was purifiedby flash column chromatography on silica gel eluting with EtOAc/Hexane(2:1) to give 2-fluoro-N-(2-hydroxyethyl)-5-nitrobenzamide 136 (17.0 g,92%) as a colorless gum. ¹HNMR [(CD₃)₂SO] δ 8.58 (br, 1H), 8.46-8.44 (m,1H), 8.41-8.31 (m, 1H), 7.60 (t, J=9.3 Hz, 1H), 4.79 (t, J=5.6 Hz, 1H),3.55-3.50 (m, 2H), 3.35-3.32 (m, 2H).

2-Fluoro-N-(2-hydroxyethyl)-5-nitrobenzamide 136 (17.0 g, 74.50 mmol)was dissolved in CH₂Cl₂ (300 mL) and treated with catalytic amount of4-methylbenzenesulfonic acid (1.28 g, 7.45 mmol) followed by3,4-dihydro-2H-pyran (13.59 mL, 149.01 mmol) at the room temperature.The reaction mixture was stirred overnight then washed with water (3×),dried with Na₂SO₄ and concentrated under reduced pressure. The crudeproduct was purified by flash column chromatography on silica geleluting with EtOAc/Hexane (1:1) to give2-fluoro-5-nitro-N-(2-((tetrahydro-2H-pyran-2-yl)oxy)ethyl) benzamide126 (16.8 g, 72%) as a colorless oil. ¹HNMR and HRMS in agreement withthe Method 1.

2-(bis(2-hydroxyethyl)amino)-5-nitro-N-(2-((tetrahydro-2H-pyran-2-yl)oxy)ethyl)benzamide (127)

2-Fluoro-5-nitro-N-(2-((tetrahydro-2H-pyran-2-yl)oxy)ethyl) benzamide126 (3.17 g, 10.15 mmol) was dissolved in dioxane (150 mL) and treatedwith Et₃N (4.24 mL, 30.45 mmol) and diethanolamine (3.89 mL, 40.60mmol). The reaction mixture was heated to 55° C. overnight then cooledto the room temperature, and the solvent was evaporated. The residue wasdissolved in EtOAc, washed with water (3×) and brine, dried with Na₂SO₄and concentrated under reduced pressure. The crude product was purifiedby flash column chromatography on silica gel eluting with EtOAc/Hexane(3:1) to give2-(bis(2-hydroxyethyl)amino)-5-nitro-N-(2-((tetrahydro-2H-pyran-2-yl)oxy)ethyl)benzamide 127 (3.51 g, 87%) as a yellow gum. ¹HNMR [(CD₃)₂SO] δ 8.71 (t,J=5.5 Hz, 1H), 8.10-8.06 (m, 2H), 7.17 (d, J=9.0 Hz, 1H), 4.73 (t, J=5.3Hz, 2H), 4.62-4.60 (m, 1H), 3.80-3.72 (m, 2H), 3.57-3.49 (m, 5H),3.47-3.40 (m, 7H), 1.77-1.70 (m, 1H), 1.66-1.61 (m, 1H), 1.50-1.46 (m,4H).

((4-nitro-2-((2-((tetrahydro-2H-pyran-2-yl)oxy)ethyl)carbamoyl)phenyl)azanediyl)bis(ethane-2,1-diyl) dimethanesulfonate (128)

A solution of2-(bis(2-hydroxyethyl)amino)-5-nitro-N-(2-((tetrahydro-2H-pyran-2-yl)oxy)ethyl)benzamide 127 (6.01 g, 15.12 mmol) in CH₂Cl₂ (180 mL) was cooled to 0°C. and treated with Et₃N (7.38 mL, 52.93 mmol) followed bymethanesulfonyl chloride (4.40 mL, 45.36 mmol). The reaction mixture waswarm to the room temperature for 30 min, washed with water (3×), driedwith Na₂SO₄ and concentrated under reduced pressure. The residues waspurified by flash column chromatography on silica gel eluting withCH₂Cl₂/MeOH (20:1) to give((4-nitro-2-((2-((tetrahydro-2H-pyran-2-yl)oxy)ethyl)carbamoyl)phenyl)azanediyl)bis(ethane-2,1-diyl)dimethanesulfonate 128 (8.04 g, 96%) as a yellow gum. 1HNMR [(CD₃)₂SO] δ8.73 (t, J=5.5 Hz, 1H), 8.15-8.10 (m, 2H), 7.27 (d, J=9.2 Hz, 1H), 4.32(t, J=5.3 Hz, 4H), 3.82-3.74 (m, 6H), 3.56-3.40 (m, 5H), 3.13 (s, 6H),1.76-1.71 (m, 1H), 1.68-1.61 (m, 1H), 1.50-1.47 (m, 4H).

((2-((2-hydroxyethyl)carbamoyl)-4-nitrophenyl)azanediyl)bis(ethane-2,1-diyl)dimethanesulfonate (129)

((4-Nitro-2-((2-((tetrahydro-2H-pyran-2-yl)oxy)ethyl)carbamoyl)phenyl)azanediyl)bis(ethane-2,1-diyl)dimethanesulfonate 128 (3.03 g, 5.47 mmol) in dry MeOH (100 mL) wastreated with methanesulfonic acid (17.8 mL, 20.37 mmol). The reactionmixture was stirred at room temperature for 20 min and the solvent wasevaporated. The residue was dissolved in EtOAc, washed with water (3×),dried with Na₂SO₄ and concentrated under reduced pressure and the crudeproduct was purified by flash column chromatography on silica geleluting with CH₂Cl₂/MeOH (19:1) to give((2-((2-hydroxyethyl)carbamoyl)-4-nitrophenyl)azanediyl)bis(ethane-2,1-diyl)dimethanesulfonate 129 (1.92 g, 75%) as a yellow gum. 1HNMR [(CD₃)₂SO] δ8.73 (t, J=5.5 Hz, 1H), 8.15-8.10 (m, 2H), 7.27 (d, J=9.2 Hz, 1H), 4.32(t, J=5.3 Hz, 4H), 3.82-3.74 (m, 6H), 3.56-3.40 (m, 5H), 3.13 (s, 6H),1.76-1.71 (m, 1H), 1.68-1.61 (m, 1H), 1.50-1.47 (m, 4H). HRMS(ESI) calcdfor C₁₅H₂₃N₃NaO₁₀S₂[M+Na]⁺ m/z 492.0717: found 492.0720.

2-((2-bromoethyl)(2-((2-hydroxyethyl)carbamoyl)-4-nitrophenyl)amino)ethylmethanesulfonate (64)

((2-((2-Hydroxyethyl)carbamoyl)-4-nitrophenyl)azanediyl)bis(ethane-2,1-diyl)dimethanesulfonate 129 (8.00 g, 17.04 mmol) was dissolved in acetone(240 mL) and treated with LiBr (1.48 g, 17.04 mmol) at the roomtemperature. The reaction mixture was stirred overnight and the solventwas removed. The residue was dissolved in EtOAc and washed with water(2×), dried with Na₂SO₄ and concentrated under reduced pressure. Thecrude product was purified by flash column chromatography on silica geleluting with CH₂Cl₂/MeOH (20:1) to give2-((2-bromoethyl)(2-((2-hydroxyethyl)carbamoyl)-4-nitrophenyl)amino)ethylmethanesulfonate 64 (3.74 g, 48%) as a yellow gum. ¹HNMR [(CD₃)₂SO] δ8.65 (t, J=5.6 Hz, 1H), 8.14-8.09 (m, 2H), 7.22 (d, J=9.1 Hz, 1H), 4.75(br, 1H), 4.31 (t, J=5.4 Hz, 2H), 3.80-3.74 (m, 5H), 3.65-3.53 (m, 5H),3.13 (s, 3H). HRMS(ESI) calcd for C₁₄H₂₁BrN₃O₇S [M+H]⁺ m/z 454.0278:found 454.0273.

2-((2-bromoethyl)(4-nitro-2-((2-(phosphonooxy)ethyl)carbamoyl)phenyl)amino)ethylmethanesulfonate (60)2-((2-bromoethyl)(2-((2-((di-tert-butoxyphosphoryl)oxy)ethyl)carbamoyl)-4-nitrophenyl)amino)ethylmethanesulfonate (130)

A stirred solution of2-((2-bromoethyl)(2-((2-hydroxyethyl)carbamoyl)-4-nitrophenyl)amino)ethylmethanesulfonate 64 (2.61 g, 5.76 mmol) in DMF (2 mL) at 10° C. wastreated with 1H-tetrazole (61.9 mL, 26.50 mmol; 3% w/w solution inMeCN), followed by the slow addition of di-tert-butyldiisopropylphosphoramidate (95%, 7.68 mL, 23.05 mmol). The mixture wasstirred at room temperature for 4 h, then cooled to 5° C. and treatedwith portionwise addition of m-CPBA (70%, 7.46 g, 43.21 mmol). Afterfurther stirring at room temperature for 2 h the reaction mixture wasconcentrated under reduce pressure and the residue was dissolved inEtOAc, washed with 10% aqueous Na₂S₂O₅, 5% aqueous NaHCO₃ and waterbefore being dried with Na₂SO₄ and concentrated under reduced pressure.The crude product was purified by flash column chromatography on silicagel eluting with EtOAc/Hexane (1:1) to give2-((2-bromoethyl)(2-((2-((di-tert-butoxyphosphoryl)oxy)ethyl)carbamoyl)-4-nitrophenyl)amino)ethylmethanesulfonate 130 (1.45 g, 39%) as a yellow gum. ¹HNMR [(CD₃)₂SO] δ8.84 (t, J=5.6 Hz, 1H), 8.13 (2d, J=2.8 Hz, 1H), 8.09 (d, J=2.8 Hz, 1H),7.23 (d, J=9.3 Hz, 1H), 4.32 (t, J=5.4 Hz, 2H), 4.00 (q, J=6.3 Hz, 2H),3.80-3.74 (m, 4H), 3.64 (t, J=6.7 Hz, 2H), 3.51 (q, J=5.6 Hz, 2H), 3.14(s, 3H), 1.42 (s, 18H). HRMS(ESI) calcd for C₂₂H₃₇BrKN₃O₁₀PS [M+K]+m/z684.0752: found 684.0740.

2-((2-bromoethyl)(4-nitro-2-((2-(phosphonooxy)ethyl)carbamoyl)phenyl)amino)ethylmethanesulfonate (60)

A stirred solution of2-((2-bromoethyl)(2-((2-((di-tert-butoxyphosphoryl)oxy)ethyl)carbamoyl)-4-nitrophenyl)amino)ethylmethanesulfonate 130 (1.45 g, 2.24 mmol) in CH₂Cl₂ (30 mL) was treatedwith TFA (10 mL) at room temperature with stirring for 1 h, thenconcentrated under reduced pressure to remove the excess TFA. Theresulted yellow residue was dissolved in EtOAc and evaporated to drynessto give2-((2-bromoethyl)(4-nitro-2-((2-(phosphonooxy)ethyl)carbamoyl)phenyl)amino)ethylmethanesulfonate 60 (1.20 g, 100%) as a yellow gum. ¹HNMR [(CD₃)₂SO] δ8.86 (t, J=5.6 Hz, 1H), 8.15-8.11 (m, 2H), 7.24 (d, J=9 Hz, 1H), 4.32(t, J=5.4 Hz, 2H), 4.07-4.02 (m, 2H), 3.79-3.74 (m, 4H), 3.65 (t, J=6.7Hz, 2H), 3.51 (q, J=5.5 Hz, 2H), 3.13 (s, 3H). HRMS(ESI) calcd forC₁₄H₂₁BrKN₃O₁₀PS [M+K]+m/z 571.9500: found 571.9451.

Example 2 Prodrug Cytotoxicity Screening

Compounds were subjected to testing, including low-cell densitycytotoxicity assay. Cells (500 cells/well) were seeded in 96-well platesand exposed to prodrug for 4 h, washed, then left to grow for a further5 days. The SRB assay was employed to determine IC50 values(concentration of prodrug required to inhibit proliferation by 50%).Parental HCT116 cells (HCT116 is a cell line derived from human coloncancer) and cells expressing the example nitroreductase gene nfsA fromEscherichia coli were evaluated head-to-head. Parental H1299 cells(H1299 is a cell line derived from lung cancer) and cells expressing theexample nitroreductase gene nfsA from Escherichia coli were evaluatedhead-to-head. Parental HCT116 cells and cells expressing human AKR1C3were evaluated head-to-head. Parental H1299 cells and cells expressinghuman AKR1C3 were evaluated head-to-head. In a variation of these assaystest compounds were assessed for their ability to give hypoxia-selectivecell killing. Here wild type HCT116, H460 (H460 is a cell line derivedfrom lung cancer), H1299 and SiHa cells (SiHa is a cell line derivedfrom cervical cancer) were used in addition to HCT116 cells engineeredto over express cytochrome P450 reductase (HCT116 POR), a humanone-electron nitroreductase. Low-cell density cytotoxicity assays wereperformed as above under oxic conditions and compared to experimentswhere cells were shown 4 h of anoxia during the prodrug exposure period,before being washed free of prodrug and then left to grow for a further5 days under oxic conditions, as above.

In parallel, a three dimensional high-cell density multi-cellular layer(MCL) clonogenic assay was performed. 1×10⁶ cells of either 100%HCT-116^(WT) or containing 97% WT with a minor 3% HCT-116^(NTR) cellpopulation were seeded into a collagen-coated Teflon microporousmembrane and left to grow for 3 days. MCLs were then exposed to prodrugfor 5 h. After treatment, MCLs were enzyme dissociated, diluted in freshmedium and plated to determine clonogenic survival. To discriminateclonogenic activator (NTR+) from target (NTR−) colonies, cells wereplated in non-selective medium (total cells) and medium containing 1 μMpuromycin (activator cells). Colonies were grown for 10 days beforestaining. Colonies containing >50 cells were counted. A variation ofthis assay was also used to assess for evidence of AKR1C3-dependentcytotoxicity. Here either 100% HCT-116W^(T) or 100% HCT-116^(AKR1C3)cells were seeded to provide MCLs that were then exposed to prodrug.

Results of the screening assays are provided in FIGS. 18A, 18B, 19A,20A, 20B, 21 and 23 and show that the compounds of the present inventionappear to be resistant to metabolism by AKR1C3 as indicated by aninability to provide increased cytotoxicity in low cell densitycytotoxicity testing in HCT116 and H1299 cells engineered to overexpress AKR1C3 when compared to the cytotoxicity of the test compoundsin the parental wild type cell lines (FIGS. 18A and 18B, respectively).As the inventors have determined that ‘false negatives’ can occur inthis assay due to metabolite diffusion into essentially infinitedilution of the assay media, the lack of AKR1C3-mediated cytotoxicitywas confirmed for all of the compounds of the present invention bycomparing clonogenic cell kill of wild type HCT116 cells and AKR1C3 overexpressing HCT116 cells grown as multicellular layers (MCLs) and exposedto test compounds. Here all test compounds were shown to give noadditional cell kill in MCLs that over express AKR1C3 compared to thewild type isogenic cell line, indicating they are not metabolised tocytotoxic metabolites by AKR1C3 (FIG. 19A).

The compounds of the present invention were shown to provide excellentbacterial nitroreductase mediated cell killing in low cell densitycytotoxicity assays, when assayed in HCT116 and H1299 cells engineeredto over express the example nitroreductase gene nfsA from Escherichiacoli, compared to the parental wild type cell lines (FIGS. 20A and 20B,respectively). Here an additional 2 to 3 logs of cell kill is observedin cells that express E coli nfsA. To confirm the test compounds providebystander cell killing, they were assessed in a three dimension highcell density assay employing mixed HCT116 MCLs that contain 97%wild-type HCT116 cells (target cells) and 3% HCT116 cells overexpressing E coli nfsA (activator cells). All compounds demonstrated theability to be metabolized by the 3% HCT116 cells over expressing E colinfsA to produce cytotoxic metabolites capable of diffusing to kill theneighbouring wild type HCT116 cells (FIG. 21).

The compounds of the present invention were tested in low cell densitycytotoxicity assays under oxic and anoxic conditions, for their abilityto give hypoxia-dependent cytotoxicity. Wild type HCT116, H460, H1299and SiHa cells were used in addition to HCT116 cells engineered to overexpress cytochrome P450 reductase (HCT116 POR), a human one-electronnitroreductase. Compounds 14, 22, 18 and 301 were shown to provideincreased cytotoxicity in cells under hypoxia. The degree of this effectwas increased for compounds 14 and 22 when HCT116 cells over expresscytochrome P450 reductase, indicating increased hypoxia-selectiveprodrug metabolism by this human nitroreductase is providing increasedcytotoxicity (FIG. 23).

Example 3 Screening of Prodrug Compounds Using Nitroreductase Library

A phylogenetically diverse library of 55 nitroreductase candidates from20 bacterial species, representing 12 different enzyme families wasscreened for their ability to co-metabolise target prodrugs.

The method employed the over-expression of a candidate nitroreductasegene from plasmid pUCX in an SOS reporter strain, as first described inProsser et al., 2010, Biochem Pharmacol 79, 678-687. In order to enhancethe sensitivity of the SOS reporter system, an sfiA::GFP reporterconstruct was integrated into a CDF-based plasmid (which contains acompatible origin of replication with pUCX) to give the pANODuetreporter plasmid for GFP screening. In addition, nfsA, nfsB, azoR, andnemA genes were deleted to minimise background metabolism, and the tolCgene deleted to minimise efflux of test compounds; this strain wasdesignated SOS-R3. Further improvements were obtained by deleting themdaB, ycaK, and yieF genes to minimise background metabolism, and byintroducing transcriptional terminators to the pANODUET reporterplasmid. This final reporter strain was designated SOS-R4.

Example 4 In Vivo Assessment of Prodrug Efficacy

Animal Husbandry

Specific pathogen-free female homozygous nude NIH-III (NIH-Lyst^(bg)Foxn1^(nu) Btk^(xid)) mice were bred by the Vernon Jansen Unit (sharedvivarium, University of Auckland). Animals were housed in Techniplastmicroisolator cages and provided with a standard twelve hour day-nightlight schedule. Animals received standard rodent diet (Harlan Tekladdiet 2018i) and water ad libitum. All animal studies were approved bythe University of Auckland Animal Ethics Committee.

Tumour Cell Inoculation

Animals weighed 18-25 g at the time of tumour inoculation. Tumours weregrown subcutaneously on the right flank of mice by inoculating cellsgrown in tissue culture (1×10⁷ cells in 100 uL serum free α-MEM). Tumoursizes were monitored three times weekly using electronic callipers andtreatments were initiated once tumour diameter reached ≥7 mm.

Growth Delay

Tumour bearing mice were randomised into the appropriate treatmentgroups and tumour size and body weight recorded. Test compounds wereformulated on the day of the experiment and kept in foil-wrapped tubesout of direct fluorescent light. If recruitment of animals occurred overmultiple days, the drug stocks were aliquoted into tubes and frozen onceat −4° C. until required. Mice were treated with a single (or BID) doseof prodrug by intraperitoneal injection and thereafter tumour size andbody weight was monitored every second day. Tumour volume was calculatedas π (l×w×w)/6, where l is the major axis and w is the perpendicularminor axis. Animals were culled when they had reached the appropriatesurvival endpoint or when body weight loss exceeded 20% of thepre-treatment value.

Excision Assay with and without Radiation

Tumours were grown subcutaneously in the flank of NIH-III mice byinoculating cells grown in tissue culture. Tumours were monitored usingelectronic calipers. When tumours reached treatment size, mice wererandomized to treatment groups (five to seven per group). Compounds weregiven as single (or BID) intraperitoneal doses alone or 5 min afterwhole body irradiation (⁶⁰Co source). Eighteen hours after treatment,tumours were excised, weighed, minced, dissociated enzymatically, andplated to determine clonogenicity. Clonogens/gram of tissue werecalculated relative to controls and effects of treatment were tested forsignificance (ANOVA with Dunnett's).

Results of the in vivo efficacy testing of prodrugs 10, 11, 22, 23, 26and 60 of the present invention in mixed HCT116 and H1299 tumourxenografts grown to contain 15% E coli NfsA expressing cells and 85%wild type cells in female NIH-Ill mice are shown in FIGS. 22A, 22B, 22C,22D, 22E and 22F. All compounds tested provide a profound tumour growthdelay following a single (or BID) intraperitioneal dose, indicatingprodrug metabolism by E coli NfsA followed by metabolite diffusion toprovide bystander cell killing in vivo.

Results of the in vivo efficacy testing of prodrugs 11, 22, 23 and 300in HCT116 POR and wild type SiHa tumour xenografts are shown in FIGS.24, 25 and 26. Here test compounds were assessed as single agents or incombination with 10 or 15Gy of radiation, following a single (or BID)intraperitioneal dose. All of the prodrugs investigated demonstratedincreased cell killing above what could be achieved with radiationalone, indicating the ability of the compounds to kill hypoxic (andtherefore radiation resistant) cells in the tumour xenograft.

Materials and Methods

Nitroreductase Gene Library

The full list of candidate genes in the 55-membered candidatenitroreductase library is as follows (ordered alphabetically by thebacterial strain (underlined) that each was amplified from): Bacilluscoaqulans (36D1) nfsA; Bacillus subtilis (ATCC 6051) nfrA, ycnD, ydgI,yfkO, ywrO; Bacillus thurinqiensis (serovar konkukian, strain 97-27)nfsA; Citrobacter koseri (ATCC 27156) nfsA, nfsB; Enterobacter(Chronobacter) sakazakii (ATCC 29544) nfsA, nfsB; Erwinia carotovora(subspecies Atrosepticum SCRI1043) nfsA; Escherichia coli (W3110) azoR,kefF, mdaB, nemA, nfsA, nfsB, wrbA, ycdI, ydjA, yieF; Klebsiellapneumoniae (ATCC 13883) nemA, nfsA, nfsB, ycdI, ydjA; Lactobacillussakei (subspecies sakei 23K) nfsA; Listeria welshimeri (serovar 6b,strain SLCC5334) nfsA; Listeria innocua (Clip11262) nfsA, ywrO;Mycobacterium smegmatis (strain MC²155) nfsA; Nostoc punctiforme (PCC73102) nfsA; Pseudomonas aeruginosa (PAO1) nfsB (PA5190), nqo1 (PA4975),yieF (PA1204); Pseudomonas putida (KT2440) azoR (PP4538), nfsA (PP2490),nfsB (PP2432), nqo1 (PP3720); Pseudomonas syringae pv. phaseolicola(1448a) mdaB, wrbA; Salmonella typhi (ATCC 19430) azoR, nemA, nfsA,nfsB; Vibrio fischeri (ATCC 7744) FRaseI (flavin reductase 1), nfsA,ywrO; Vibrio harveyi (ATCC 33843) frp (flavin reductase P), nfsB; Vibrioharveyi (HY01) CO-frp (E. coli codon optimized variant of flavinreductase P); Vibrio vulnificus (ATCC 27562) azoR, nfsA, nfsB, nemA. Todistinguish genes or enzymes with the same family name, for the purposeof this work each oxidoreductase was referred to using standardnomenclature followed by an underscore and a two letter abbreviation ofthe genus and species, e.g. NfsA_Kp and NemA_Ec for the NfsA enzyme fromK. pneumoniae and NemA enzyme from E. coli, respectively.

In addition to screening the 55-membered candidate nitroreductaselibrary, activity with compounds 14, 18, 22, 23, 24, 25, 26, 27 and 64was demonstrated for a range of single and poly mutated variants of E.coli NfsA (NfsA_Ec), and a single-mutated variant of B. subtilis NfrA(NfrA_Bs/NfsA_Bs) that had previously been engineered for enhancedactivity with PR-104A. The single-mutated variants of NfsA_Ec wereNfsA_Ec_12S (native arginine at position 12 substituted by serine);NfsA_Ec_41Y (native serine at position 41 substituted by tyrosine);NfsA_Ec_134A (native asparagine at position 134 substituted by alanine);and NfsA_Ec_222E (native lysine at position 222 substituted byglutamate). The single-mutated variant of NfsA_Bs was NfsA_Bs_234P(native arginine at position 234 substituted by proline). Thepoly-mutated variants of E. coli NfsA were poly17 (I5T, S41Y, R225P,F227S), poly22 (S41Y, E99G, L103M, R225P, F227S) and poly42 (S41Y,L103M, R225G, F227S).

GFP-SOS Assays

Stored glycerols of the 55-membered nitroreductase library in SOS-R4were thawed and used to inoculate overnight cultures (Lysogeny Broth(LB) amended with 100 μg ml⁻¹ Ampicillin, 50 μg ml⁻¹ Spectinomycin, 0.4%glucose) that were incubated at 30° C., 200 rpm for 16 h. The nextmorning the GFP-SOS assay was commenced by inoculation of 195 μl freshassay media (LB+100 μg ml⁻¹ Ampicillin, 50 μg ml⁻¹ Spectinomycin, 0.2%glucose, 50 μM IPTG) with 15 μl of overnight culture in individual wellsof a 96-well plate. Plates were incubated at 30° C., 200 rpm for 2.5 h(pre-challenge period), following which cultures were diluted 1:2 bysplitting 50:50 into fresh assay media (+DMSO to 0.5% finalconcentration) and fresh challenge media (assay media+drug to desiredconcentration, DMSO to 0.5% final concentration) to final volumes of 80μl apiece in duplicate on a 384-well plate. Plates were incubated at 30°C., 200 rpm for 6 h (challenge period). GFP expression was determined atexcitation 488 nm/emission 509 nm. Turbidity was determined by OD₆₀₀ andfluorescence data from any replicates not found to be within 15% of themedian culture turbidity were discarded.

Purified Enzyme Kinetics

For compounds 14, 18, 22, 23, 24, 25, 26, 27 and 64 steady-state enzymekinetics with purified NfsA_Ec were assessed spectrophotometrically at400 nm to directly monitor compound reduction, as described for PR-104Ain Prosser et al. (2013). Molar extinction coefficients of 4800 M⁻¹ cm⁻¹(compounds 14 and 18), 5200 M⁻¹ cm⁻¹ (compound 22), 5500 M⁻¹ cm⁻¹(compounds 25 and 26), 5600 M⁻¹ cm⁻¹ (compounds 23, 24 and 27) and10,000 M⁻¹ cm⁻¹ (compound 64) were measured (as described for PR-104A inProsser et al., 2013, Biochem Pharmacol 85, 1091-1103) and used for thecalculation of enzyme activity. Reactions were performed in 60 μl inUVettes (Eppendorf), using the 2 mm light path length. Reactionscontained 10 mM Tris-Cl (pH 7.0), 4% DMSO, 0.25 mM NADPH and varyingcompound concentrations. Reactions were initiated by addition of 6 μlenzyme and changes in absorbance were measured for 20 s (duringlinearity). For calculation of K_(m) and k_(cat), substrateconcentrations were varied from ˜0.2×K_(m) up to either 5×K_(m) or themaximum concentration permitted by compound solubility. Non-linearregression analysis and Michaelis-Menten curve fitting was performedusing Sigmaplot 10.0 (Systat Software Inc.).

NfsB Rate Assay

The solubility limits of the compounds meant that apparent K_(m) andk_(cat) parameters could not be accurately determined for NfsB_Ec, whichexhibited a linear rate of reduction for all compounds within theachievable concentration ranges. Instead, assessment of the relativerate of compound reduction by NfsB_Ec at one fixed concentration (600μM) of each compound was performed. Individual reactions wereestablished in an identical manner to that described for determinationof purified enzyme kinetics for NfsA_Ec.

Results

Results of the NTR screening of the prodrugs in bacteria and the NfsAkinetics and NfsB rate of metabolism assays are provided in FIGS. 17B to17L. Compounds 14, 18, 22, 23, 24, 25, 26, 27 and 64 were all confirmedto be substrates for multiple wild type and mutant bacterialnitroreductases from the NfsA and NfsB families from several species ofbacteria, as assessed by their ability to be metabolised by thebacterial nitroreductase to genotoxic metabolites that result in an SOSresponse in bacteria (FIG. 17B to 17J). Metabolism of the test compoundsby E coli NfsA was confirmed by incubating varying concentrations of thecompounds in the presence of recombinant E coli NfsA and NADPH co-factorand then following the loss of co-factor over time. Enzyme K_(m)'s wereconfirmed to range from 160 to 820 uM. Enzyme k_(cat) ranged from 3.6 to21.9 s⁻¹ (FIG. 17K). Metabolism of the test compounds by E coli NfsB wasconfirmed by incubating one fixed concentration (600 μM) of eachcompound in the presence of recombinant E coli NfsB and NADPH co-factorand then following the loss of co-factor over time. All compounds testedwere metabolised by E coli NfsB with rates varying from 200 to 4500umol/min/mg (FIG. 17L).

Throughout this specification, unless the context requires otherwise,the words “comprise”, “comprising” and the like, are construed in aninclusive sense as opposed to an exclusive sense, that is to say, in thesense of “including, but not limited to”.

The reference to any prior art in this specification is not, and shouldnot be taken as, an acknowledgement or any form of suggestion that thatprior art forms part of the common general knowledge.

It will be appreciated that the compounds of the invention may occur indifferent geometric and enantiomeric forms, and that both pure forms andmixtures of these compounds are included.

The entire disclosures of all applications, patents and publicationscited above and below, if any, are herein incorporated by reference.

The invention may be said broadly to consist in the parts, elements andfeatures referred to or indicated in the specification, individually orcollectively, in any or all combinations of two or more of said parts,elements or features.

Wherein the foregoing description reference has been made to integers orcomponents having known equivalents thereof, those integers are hereinincorporated as if individually set forth.

It should be noted that various changes and modifications to thepresently preferred embodiments described herein will be apparent tothose skilled in the art. Such changes and modifications may be madewithout departing from the spirit and scope of the invention and withoutdiminishing its attendant advantages. It is therefore intended that suchchanges and modifications be included within the scope of the invention.

The invention claimed is:
 1. A method for treating cancer, the methodcomprising administering a compound of formula (I), or apharmaceutically acceptable salt thereof, in a therapeutically effectiveamount to a tumor cell, or therapeutically proximate to said tumor cell,in a solid and/or hypoxic tumor in a subject, wherein the cancer isselected from colon cancer, cervical cancer, or lung cancer:

wherein W represents Cl, Br, I, OSO₂R, X represents Cl, Br, I, OSO₂R, Yrepresents H, CN, SO₂R, each R independently represents a lower C₁₋₆alkyl group, Z is selected from any of the radicals

wherein R₁ represents H, or a lower C₁₋₆ alkyl group; R₂ represents H,or a lower C₁₋₆ alkyl group; n represents 2 to 6; * represents a pointof attachment to formula (I); or a pharmaceutically acceptable salt ofsaid compound.
 2. The method according to claim 1, wherein the compoundof formula (I) is represented by the formula (Ih)

wherein W represents Cl, Br, I, OSO₂R, X represents Cl, Br, I, OSO₂R, Rindependently represents a lower C₁₋₆ alkyl group, and R₁ represents H,or a lower C₁₋₆ alkyl group.
 3. The method according to claim 2, whereinthe compound of formula (Ih) is represented by the following formula,


4. The method according to claim 1, wherein the compound is selectedfrom the group consisting of:(5-(bis(2-bromoethyl)amino)-4-(methylsulfonyl)-2-nitrophenyl)(4-methylpiperazin-1-yl)methanone(compound 22),(5-(bis(2-bromoethyl)amino)-4-(methylsulfonyl)-2-nitrophenyl)(4-ethylpiperazin-1-yl)methanone (compound 23), (5-(bis(2-bromoethyl)amino)-4-(methylsulfonyl)-2-nitrophenyl)(4-isopropylpiperazin-1-yl)methanone(compound 24), (5-(bis(2-bromoethyl)amino)-4-(ethylsulfonyl)-2-nitrophenyl)(4-methylpiperazin-1-yl)methanone(compound (25), (5-(bis(2-bromoethyl)amino)-4-(ethylsulfonyl)-2-nitrophenyl)(4-ethylpiperazin-1-yl)methanone (compound26), (5-(bis(2-bromoethyl)amino)-4-(ethylsulfonyl)-2-nitrophenyl)(4-isopropylpiperazin-1-yl)methanone(compound 27),2-((2-bromoethyl)(5-(4-methylpiperazine-1-carbonyl)-2-(methylsulfonyl)-4-nitrophenyl)amino)ethylmethanesulfonate (compound 310), 2-((2-bromoethyl)(5-(4-ethylpiperazine-1-carbonyl)-2-(methylsulfonyl)-4-nitrophenyl)amino)ethylmethanesulfonate (compound 311), 2-((2-bromoethyl)(5-(4-isopropylpiperazine-1-carbonyl)-2-(methylsulfonyl)-4-nitrophenyl)amino)ethylmethanesulfonate (compound 312),2-((2-bromoethyl)(2-ethylsulfonyl)-5-(4-methylpiperazine-1-carbonyl)-4-nitrophenyl)amino)ethylmethanesulfonate (compound 313),2-((2-bromoethyl)(5-(4-ethylpiperazine-1-carbonyl)-2-(ethylsulfonyl)-4-nitrophenyl)amino)ethylmethanesulfonate (compound 314), and2-((2-bromoethyl)(2-ethylsulfonyl)-5-(4-isopropylpiperazine-1-carbonyl)-4-nitrophenyl)amino)ethylmethanesulfonate (compound 315).
 5. The method according to claim 1,wherein the compound is a pharmaceutically acceptable salt of saidcompound and the salt is a methanesulfonate salt.
 6. The methodaccording to claim 1, wherein the compound is a methanesulfonate salt of((2 bromoethyl)(5 (4 ethylpiperazine 1 carbonyl) 2(methylsulfonyl)-4nitrophenyl)amino)ethyl methanesulfonate (compound 311).
 7. The methodaccording to claim 1, wherein the tumour cells express nitroreductase.8. The method according to claim 1, further including the steps of a)irradiating the tumour cells; or b) administering one or morechemotherapeutic agents and/or therapies to the subject; before, duringor after the administration of the compounds of formula (I).
 9. Themethod according to claim 1, wherein the compound is administered to asubject in combination with a) at least one nitroreductase enzymecapable of metabolising the compound; or b) a therapy that results inexpression of an exogenous nitroreductase enzyme within, ortherapeutically proximate to, a tumour.
 10. The method according toclaim 1, wherein said therapeutically effective amount of a compound offormula (I) is comprised in a pharmaceutical composition furthercomprising at least one of a pharmaceutically acceptable excipient,adjuvant, carrier, buffer or stabiliser.
 11. The method according toclaim 1, wherein the method further comprises dissolving the compound offormula (I) in an aqueous solution.
 12. The method according to claim 1,wherein said composition is formulated for parenteral administration.13. The method according to claim 1, wherein said composition is in theform of a tablet, capsule, powder, or liquid.
 14. A method for treatingcancer or a hyperproliferative condition in a subject in need thereof,the method comprising administering a therapeutically effective amountof the methanesulfonate salt of ((2 bromoethyl)(5 (4 ethylpiperazine 1carbonyl) 2(methylsulfonyl)-4nitrophenyl)amino)ethyl methanesulfonate toa tumor cell, or therapeutically proximate to said tumor cell, in asolid and/or hypoxic tumor in the subject.
 15. The method according toclaim 14, wherein the tumour cells express nitroreductase.
 16. Themethod according to claim 14, further including the steps of a)irradiating the tumour cells; or b) administering one or morechemotherapeutic agents and/or therapies to the subject; before, duringor after the administration of the compounds of formula (I).
 17. Themethod according to claim 14, wherein the compound is administered to asubject in combination with a) at least one nitroreductase enzymecapable of metabolising the compound; or b) a therapy that results inexpression of an exogenous nitroreductase enzyme within, ortherapeutically proximate to, a tumour.
 18. The method according toclaim 14, wherein said therapeutically effective amount of a compound offormula (I) is comprised in a pharmaceutical composition furthercomprising at least one of a pharmaceutically acceptable excipient,adjuvant, carrier, buffer or stabiliser.
 19. The method according toclaim 14, wherein the method further comprises dissolving the compoundof formula (I) in an aqueous solution.
 20. The method according to claim14, wherein said composition is formulated for parenteraladministration.
 21. The method according to claim 14, wherein the canceris selected from colon cancer, cervical cancer, or lung cancer.